©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Expression of the Rat Testis-specific Histone H1t Gene in Transgenic Mice
ONE KILOBASE OF 5`-FLANKING SEQUENCE MEDIATES CORRECT EXPRESSION OF A lacZ FUSION GENE (*)

(Received for publication, September 1, 1995; and in revised form, October 31, 1995)

John G. Bartell (§) Tia Davis (1) Eric J. Kremer (¶) Michael J. Dewey (1) W. Stephen Kistler (**)

From the Department of Chemistry and Biochemistry, School of Medicine Department of Biological Sciences, University of South Carolina, Columbia, South Carolina 29208

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

H1t is synthesized in mid to late pachytene spermatocytes of the male germ line and is the only tissue-specific member of the mammalian H1 histone family. As a step toward identifying DNA sequences that confer its tissue-specific expression, we have produced transgenic mice containing the intact rat H1t gene as well as a H1t-lacZ fusion gene. Transgenic mice carrying a 6.8-kilobase fragment of rat genomic DNA encompassing the H1t gene expressed rat H1t at high levels in the testis and in no other organ examined. H1t fragments truncated to within 141 base pairs (bp) of the gene in the 5` direction or within 837 bp in the 3` direction retained testis specificity. Expression of rat H1t protein was also evident in the testes of the transgenic mice, and in some lines the level of rat H1t exceeded that of the mouse protein. The stage of spermatogenesis of transgene expression was assessed by following appearance of transgenic mRNA in developing mice and by immunohistochemistry using an antiserum to rat H1t. In lines from three different constructs, expression was restricted to germinal cells, although in two strongly expressing lines the transgenes were expressed somewhat prematurely in preleptotene spermatocytes. An H1t(-948/+71)-lacZ fusion was also expressed specifically in the spermatocytes and round spermatids of a transgenic line, confirming that sequences sufficient for correct tissue and developmental expression lie within this 1,019-bp segment of the gene.


INTRODUCTION

Spermatogenesis involves a complex developmental program in which relatively undifferentiated cells of the male germ lineage transform into spermatozoa. A substantial list of testis-specific genes that are expressed in a particular temporal order during mammalian spermatogenesis is known (reviewed in Wolgemuth and Watrin(1991) and Hecht(1993)), and a current challenge is to understand the factors responsible for this pattern of gene regulation.

The testis-specific histone variants are particularly interesting because they belong to families that each have many members expressed in somatic cells. As the fundamental packaging proteins of chromatin (Wolffe, 1992), the five histone classes are found in all tissues. However, in mammals the male germ line is unique in having specific histone variants not expressed elsewhere. These include the variants H1t (Seyedin and Kistler, 1980; Cole et al., 1986), H2A (Trostle-Weige et al., 1982; Huh et al., 1991), and H2B (Shires et al., 1976; Kim et al., 1987), which have been characterized both as proteins and cloned genes, whereas the variant TH3 has been identified at the protein level (Trostle-Weige et al., 1984). An H4 gene linked to the H1t gene and also expressed at high levels in the testis has been designated H4t, although it encodes the standard somatic form of the H4 protein (Grimes et al., 1987).

In rats these testis histones are expressed according to different schedules, with TH3 appearing in late spermatogonia, TH2A and TH2B appearing early in cells undergoing meiosis (spermatocytes), and H1t appearing only later in mid to late pachytene spermatocytes (Bucci et al., 1982; Meistrich et al., 1985; Kremer and Kistler, 1991). These variants replace their somatic counterparts to varying degrees and remain in developing germ cells until the haploid nucleus elongates and condenses, when they disappear and are replaced by spermatid/sperm-specific nuclear proteins. Unlike the situation in various invertebrates such as sea urchins (Poccia, 1986), the testis-specific histone variants of mammals are not retained in the nuclei of mature sperm.

H1 histones are believed to be responsible for condensing adjacent nucleosomes into the compact, solenoidal, 30-nm chromatin fiber. H1t, while bearing an unmistakable resemblance to members of the common somatic H1 family, is sufficiently different in amino acid sequence to constitute a recognizable variant class (Kistler, 1989), and a recent study suggests that H1t is unable to bring about a condensed chromatin state (De Lucia et al., 1994).

Isolation of the H1t gene (Cole et al., 1986; Grimes et al., 1987; Drabent et al., 1991) revealed that its immediate 5`-flanking region was extremely similar to the promoter regions for standard H1 variants expressed in somatic cells. This similarity includes four nucleotide regions found just upstream of all standard H1 genes (Coles and Wells, 1985; Heintz, 1991). A study of the role of H1t upstream sequences fused to a reporter gene in a transfected cell line revealed some variation in expression depending on the length of upstream sequences present but could not address the issue of testis specificity (Kremer and Kistler, 1992).

Transgenic mice have proved useful in defining DNA regions necessary for testis expression of such genes as protamine 1 (Peschon et al., 1987; Zambrowicz et al., 1993), protamine 2 (Stewart et al., 1988), the testis-specific form of angiotensin-converting enzyme (Langford et al., 1991), phosphoglycerate kinase-2 (Robinson et al., 1989), and proenkephalin (Galcheva-Gargova et al., 1993). The present report describes analysis of the expression of the rat H1t gene in transgenic mice in order to establish the boundaries of the DNA region necessary for developmental and tissue-specific expression. Using genomic fragments containing the natural gene, we found that constructs with as little as 141 bp (^1)of upstream or 800 bp of downstream sequence result in testis-specific expression, although elements affecting the level of expression may lie outside this region. A construct with 0.95 kb of upstream sequence fused to the Escherichia coli lacZ gene also expressed uniquely within the germ cells of the testis. Interestingly, some transgenic mice expressed the rat gene prematurely during spermatogenesis, and in some lines the level of expression of the transgene exceeded that of the endogenous H1t gene.


EXPERIMENTAL PROCEDURES

Constructs and Transgenic Mice

Constructs were derived from a 6.8-kb EcoRI genomic fragment containing the rat H1t gene (Cole et al., 1986; Grimes et al., 1990) using standard cloning techniques (Sambrook et al., 1989) and are diagrammed in Fig. 1. The TG1 and TG2 fragments were excised directly from this clone, whereas the TG3 and TG4 fragments each required sequential transfer of selected restriction fragments to pBluescript SK(+) (Stratagene, La Jolla, CA). For the H1t-lacZ fusion construct, the +70Tth111I site in the H1t 5`-UTR was blunted with Klenow DNA polymerase and joined to an NcoI linker (5`-AGCCATGGCT-3`). A fragment running from this NcoI site upstream to the -948 PvuII site was then joined to placI, obtained from R. Palmiter. This promoterless lac expression vector has an NcoI site at the initiating ATG and is identical to placF (Mercer et al., 1991) except that the intron and poly(A) regions downstream from lacZ were provided by the mouse metallothionein II gene fragment from BamHI (+350) to PstI (+840) (Searle et al., 1984). Fragments for injection were excised from plasmid vectors, resolved by agarose gel electrophoresis, transferred electrophoretically to pieces of NA45 membrane (Schleicher & Schuell), eluted in 1.5 M NaCl, extracted with n-butanol, and ethanol precipitated. In some cases DNA fragments were recovered from agarose gels using glass beads (Qiaex, QIAGEN, Chatsworth, CA) as described by the manufacturer. DNA was then dissolved in sterile 0.1 mM EDTA, 10 mM Tris-HCl (pH 8.0) at about 2 µg/ml and injected into the pronuclei of (C57BL/6 times DBA/2)F2 embryos (Brinster et al., 1985). Transgenic founder animals were identified by Southern blotting of RsaI-digested DNA obtained from tail samples (Hogan et al., 1986). For natural gene constructs these blots were hybridized to a P-labeled PstI-SalI fragment that includes most of the rat gene and cross-hybridizes to mouse H1t (see Fig. 1, probe B). Transgenic founders were subsequently mated to pure strain C57BL/6 mice; positive offspring from this cross were likewise mated and the line thus propagated through successive generations.


Figure 1: Diagram of cloned rat genomic DNA fragments used as transgenes and probes. Nucleotides are numbered relative to the transcriptional initiation point. TG1 (-2383/+4488) is bounded by EcoRI sites. TG2 (-948/+4488) is truncated upstream at the single PvuII site. TG3 (-141/+4488) is truncated upstream at a PstI site. TG4 (-2149/+1578) is truncated upstream at a SacI site and downstream at a StuI site. TG-Lac consists of the -948/+71 fragment of H1t fused to the lacZ expression module of placI as described under ``Experimental Procedures.'' Probe A (PstI -141/PstI +90) includes the 5`-UTR as well as the promoter region. Probe B (PstI +91/SalI +804) includes all of the gene except for the 5`-UTR.



The number of copies of integrated transgenes in members of established lines was estimated by immobilizing 5 µg of genomic DNA as dots on nitrocellulose (Schleicher & Schuell, BA85) and hybridization to randomly primed P-labeled probe A (see Fig. 1). Dots were excised and counted in a scintillation counter, and cpm hybridizing to the transgene was calculated by subtracting cpm hybridizing to control mouse DNA. The cpm hybridizing to rat genomic DNA were used as a standard, assuming two copies of the gene per diploid genome.

RNA Analysis

RNA was extracted from tissues by the method of Chomczynski and Sacchi(1987). For Northern blotting, 20 µg of total RNA was analyzed on a 1.5% agarose/formaldehyde gel containing 0.5 µg/ml ethidium bromide (Davis et al., 1986). Uniformity of RNA loading was checked under UV illumination. RNA was transferred to Hybond N membranes (Amersham Corp.), immobilized by exposure to UV light, and probed with a P-labeled 231-bp PstI restriction fragment that contains the entire 69 bp of the 5`-UTR and 21 bp of the coding region of rat H1t mRNA (see Fig. 1, probe A). Hybridization was at 42 °C in 0.2% bovine serum albumin, 0.2% Ficoll, 0.2% polyvinylpyrolidone, 10% dextran sulfate, 1% SDS, 100 µg/ml sheared denatured calf thymus DNA, 5 mM EDTA, 50 mM NaH(2)PO(4) (pH 7.2), 0.9 M NaCl, 50% formamide. Final washes were in 0.1% SDS, 75 mM NaCl, 0.5 mM EDTA, 5 mM Tris-HCl (pH 8) at 68 °C. Membranes were exposed to Kodak X-Omat AR film in the presence of an intensifying screen.

For S1 nuclease mapping, the 231-bp PstI fragment (-141/+90) that overlaps the cap site (see Fig. 1, probe A) was ligated to the PstI site of M13 mp18, and a single stranded uniformly P-labeled probe was generated using Klenow DNA polymerase I with the universal M13 forward sequencing primer, followed by cleavage with EcoRI and isolation of the probe by elution from a denaturing polyacrylamide gel. RNA (10 µg) was incubated with 10^5 cpm of probe, hybridized, and digested with S1 nuclease as described (Berk, 1989). The protected fragment was analyzed on a 8% sequencing type gel, which was dried and exposed to film.

Extraction and Analysis of H1t

A mouse testis was homogenized in 5 ml of cold 0.2 M H(2)SO(4), and then brought to 3% trichloroacetic acid by addition of 0.5 ml of 33% trichloroacetic acid. After 15 min on ice, the mixture was centrifuged 10,000 times g for 15 min, and volume of 100% trichloroacetic acid was added to the supernatant. After 15 min on ice and centrifugation as before, the supernatant was discarded and the pellet was rinsed twice with ethanol:ether (1:1) and allowed to dry. The H1 proteins were dissolved in 10 mM acetic acid at 1 ml/g of tissue extracted. This extract also contains high mobility group (HMG) proteins, of which HMG 1 and 2 migrate in the vicinity of rat H1t on SDS-polyacrylamide gel electrophoresis. To eliminate HMG contamination, the H1 extract was diluted to 1 ml with 0.1 M NaH(2)PO(4) (pH 7.3) and applied to a 200-µl column of BioRex 70 (200-400 mesh, Bio-Rad) equilibrated with the same buffer in a 1-ml disposable syringe. HMGs were eluted from the column by 1 ml of 7% guanidine HCl, 0.1 M NaH(2)PO(4) (pH 7.3), and the H1 fraction was then eluted by 300 µl of 15% guanidine HCl, 0.1 M NaH(2)PO(4) (pH 7.3). The H1 fraction was desalted with a 2-ml column of Sephadex G-25 medium, developed with 10 mM acetic acid, and lyophilized. H1 proteins were analyzed on a 15% SDS-polyacrylamide gel prepared as described by Laemmli(1970) except that the stacking gel was made with the same buffer as the resolving gel. This modification enhanced the separation of mouse and rat H1t. Protein bands were visualized by staining with Coomassie Brilliant Blue R-250.

Immunohistochemistry

Testes were fixed by immersion in Bouin's solution (glacial acetic acid, formalin, saturated picric acid, 1:5:15), embedded in paraffin, sectioned, mounted, and hydrated by standard methods. Slides were immersed successively for 5 min each in 1% LiCO(3)/70% ethanol, 1% H(2)O(2)/70% ethanol, and 0.3 M glycine. Using a humidified chamber, sections were then incubated in TBS (150 mM NaCl, 20 mM Tris-HCl, pH 7.4) containing 10% normal goat serum for 15 min followed by three washes in TBST (TBS containing 0.1% Tween 20). Rabbit anti-rat H1t (^2)was diluted 1:100 in TBS containing 10% goat serum and 1 mM EDTA and incubated at 37 °C for 1.5 h. Slides were then washed three times in TBST. Peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad) diluted 1:1000 in TBS containing 10% goat serum was applied and incubated at 37 °C for 30 min. Slides were washed twice in TBST and once in TBS before developing with 0.06% diaminobenzidine, 0.03% H(2)O(2), 50 mM Tris-HCl (pH 7.6). In some cases slides were developed with the Immunopure Metal Enhanced DAB Substrate Kit (Pierce) and stained lightly with Gill's hematoxylin (Fisher). Histochemical staining for beta-galactosidase was done essentially as described by Behringer et al.(1993) using decapsulated testes with the tubules slightly teased apart to insure complete penetration by the chromogenic substrate. The use of detergents at the indicated concentrations (Behringer et al., 1993) largely suppressed endogenous beta-galactosidase activity characteristic of interstitial cells.

beta-Galactosidase Assays

Tissue extracts were prepared by disrupting tissues with a glass/glass homogenizer in 5 vol of 100 mM Na(2)HPO(4) (pH 7.3), 0.1 mM MgCl(2), 2 mM MgSO(4), 40 mM 2-mercaptoethanol, 0.2 mM phenylmethylsulfonyl fluoride, 0.005% leupeptin, and 0.1% Triton X-100. Extracts were clarified by centrifugation at 10,000 times g for 15 min at 4 °C. Assays were performed as described (Sambrook et al., 1989) except they were monitored continuously with a recording spectrophotometer at 420 nm using an extinction coefficient for o-nitrophenol of 10,650 cm^2bulletmol to compensate for incomplete dissociation of the reaction product at pH 7.3. Protein was determined by the biuret reaction.


RESULTS

Overview of Natural Transgene Constructs and Their Expression

It was possible to use the entire rat H1t gene in transgenic experiments because of its small size and because the mRNA and protein encoded can each be distinguished from the products of the endogenous mouse H1t gene. For the initial experiment, we used a 6.8-kb EcoRI genomic clone containing 2.4 kb upstream of the transcriptional initiation site and 3.8 kb of sequence downstream from the 3` end of the gene (see Fig. 1for diagram of constructs). The downstream region of this fragment (Fig. 1, TG1) also contains the H4t gene (Grimes et al., 1987, 1990). Potential transgenic animals were screened by Southern analysis of tail biopsy DNA using a 713-bp PstI-SalI restriction fragment that hybridizes to both rat and mouse H1t genes (Fig. 1, probe B) and identifies a characteristic 1.4-kb RsaI fragment of the rat gene. Two founder animals were identified for this construct, TG1-555 and TG1-688, and each was bred to develop lines. Testis RNA from each line was assayed for the presence of rat-specific H1t mRNA by both S1 nuclease assays and by Northern blotting. Both lines were found to express the transgene prominently in an organ-specific fashion by either assay. Fig. 2shows testis-specific expression of the TG1-688 line by Northern blot, and Fig. 3shows testis-specific expression of the TG1-555 line by S1 assay. Thus, by either assay strong signals were evident in RNA samples from transgenic testis and undetectable with RNA from spleen, small intestine, heart, liver, lung, kidney, or brain. The S1 assay verified that the transgene mRNA in both TG1 lines protected the same probe fragment as normal rat testis H1t mRNA, indicating that transcriptional initiation was occurring correctly (Fig. 3). In addition, the sensitivity of the S1 assay emphasized that leaky expression was very low or nonexistent in the somatic organs tested. Data for these and other transgenic animals are summarized in Table 1.


Figure 2: Transgene expression analyzed by Northern blots. RNA samples were prepared, separated, and blotted to nylon membranes as described under ``Experimental Procedures.'' Blots were hybridized to P-labeled probe A (Fig. 1), which is specific for the 5`-UTR of rat H1t mRNA. The first four lanes contain samples from nontransgenic mice (lanes 1 and 2) and rats (lanes 3 and 4) to demonstrate the specificity of the probe for rat H1t. Lanes 5-12 contain samples from tissues of individual male animals from the indicated transgenic lines.




Figure 3: Transgene expression analyzed by S1 nuclease digestion. RNA samples were prepared and hybridized to a uniformly P-labeled single stranded probe overlapping the 5` end of the rat H1t mRNA, digested with S1 nuclease, and resolved on a denaturing polyacrylamide gel as described under ``Experimental Procedures.'' Correctly initiated rat H1t mRNA protects 90 bp of this probe. Lane 1, end-labeled HinfI digest of pBR322. Lane 2, 10,000 cpm of undigested probe. RNA from nontransgenic mouse organs (lanes 3 and 4) and rat organs (lanes 5 and 6) demonstrate the specificity of the probe. (The full-length probe remaining in the rat liver sample (lane 5) was not reproducible and was apparently due to incomplete digestion of this sample.) Lanes 7-13 contain RNA from organs of a single male animal of the TG1-555 line.





Encouraged by this result, we truncated this fragment about 1 kb upstream of the gene at a PvuII site to yield the TG2 construct. Three founder animals were identified for this construct, of which two (TG2-97 and TG2-99) were bred to generate lines. Each TG2 line was found to display testis-specific expression of the transgene by both S1 assays (not shown) and Northern blots (see Fig. 2, TG2-99).

We then truncated the upstream region at a PstI site 141 bp from the cap site (Fig. 1, TG3) and also investigated the region downstream of the gene by shortening a fourth construct at a StuI site at +1578 relative to the cap site (Fig. 1, TG4). Five founder animals were identified for each of these constructs (see Table 1). Three of the TG3 constructs passed the transgene and generated lines (TG3-4301, TG3-5592, and TG3-5597), whereas two of the founders failed to generate lines. Testis-specific expression of the transgene was determined by Northern blotting for all three lines, for example TG3-4301 (Fig. 2). Lines were derived from all five of the TG4 founders. Four of them gave testis-specific expression detected either by S1 analysis or Northern blotting, for example TG4-4616 (Fig. 2). The fifth TG4 founder, TG4-308, was a male that passed the transgene to each of 15 daughters but to none of 18 sons, suggesting that the transgene had integrated on the X chromosome. His daughters passed the transgene to approximately 50% of their sons, as expected, but none of those tested expressed rat H1t. This may be explained by the general inactivity of the X chromosome during meiosis in male mammals (Gordon and Ruddle, 1981).

Comparative Expression of the Rat H1t Transgene in Testes of Different Lines

Among different lines the level of transgene expression was quite variable and presumably reflects the influence of adjacent sequences in different chromosomal integration sites. However, the tissue specificity of transgene expression was not influenced by integration site because the expression of all H1t constructs was limited to the testis. To compare the relative level of transgene expression among the lines developed from our constructs we prepared a Northern blot from representatives of the four families of transgenic lines (Fig. 4). Control RNA samples from nontransgenic mouse and rat testis served to verify specificity of the probe (Fig. 4, lanes 1 and 2). Both TG1 lines (Fig. 4, lanes 3 and 4) had higher levels of rat H1t mRNA in testicular RNA samples than did rat testis RNA itself. The single TG2 line analyzed (Fig. 4, lane 5) had somewhat lower levels of H1t mRNA, comparable with those seen in the rat testis lane. Expression among the three TG3 constructs was somewhat variable (Fig. 4, lanes 6-8), ranging from about the same as rat testis to somewhat lower. Expression among the five TG4 lines was the most variable (Fig. 4, lanes 9-13), with expression ranging from undetectable (lanes 12 and 13) to very high and comparable with the TG1 lines (lane 11). Although TG4-296 expression was not detected by this Northern blot, a low level of expression was seen by S1 nuclease assay (not shown). Expression levels among these various constructs was not strictly tied to copy number of the transgene, but the general pattern was for higher copy number lines to have higher expression, particularly when comparing members of the same construct. Thus TG3-4301 (34 copies) was stronger than TG3-5592 (9 copies) or TG3-5597 (11 copies). Among the TG4 lines, TG4-4616 (30 copies) was by far the strongest expressor, whereas TG4-286 (20 copies) and TG4-4605 (9 copies) were substantially weaker, although TG4-4604 (9 copies) and TG4-296 (7 copies) were weaker still. As mentioned above, the lack of detectable expression for TG4-308 (2 copies) may have been due to its location on the X chromosome.


Figure 4: Transgene expression in the testes of animals from different lines analyzed by Northern blotting. RNA was prepared, separated, and hybridized to uniformly P-labeled probe A (Fig. 1) as described under ``Experimental Procedures.'' The specificity of the probe is shown by its selective hybridization to rat testis (lane 2) but not mouse testis (lane 1) RNA. The remaining lanes show results from RNA samples from the testes of individual male animals of the indicated transgenic lines. Ribosomal RNA bands stained with ethidium bromide (lower panel) demonstrate lack of degradation and comparable loading of the various lanes.



Mouse and rat H1t could be resolved from one another by SDS gel electrophoresis, and it was therefore possible to examine the relative concentrations of H1t protein generated by both the endogenous mouse gene and the transgene. Protein samples were prepared from seven of the transgenic lines and examined by SDS-polyacrylamide gel electrophoresis (Fig. 5). In general, those lines with a high level of transgene mRNA also expressed high levels of rat H1t protein with a concomitant reduction of endogenous mouse H1t. In some cases the level of rat H1t exceeded that of mouse H1t (Fig. 5, TG1-688, TG2-97, and TG2-99). Line TG4-296, which did not show transgene expression by Northern blot, did show a faint level of transgene protein (Fig. 5). TG1-555 was the only exception to the generalization regarding protein expression, because this line had a high level of rat mRNA (Fig. 3, lane 3) but only a low level of protein expression (Fig. 5). This discrepancy remains unexplained.


Figure 5: Transgene expression in the testes of animals from different lines analyzed by SDS-polyacrylamide gel electrophoresis. A protein fraction consisting primarily of H1 histones was prepared from the testes of individual animals by differential trichloroacetic acid precipitation and stepwise elution from BioRex 70 as described under ``Experimental Procedures.'' This fraction (about 40 µg for the majority of samples) was separated on a 15% SDS gel and stained with Coomassie Blue. The lanes for rat and mouse show samples from control animals. Rat H1t, which migrates slightly more rapidly than mouse H1t, is indicated by the pointer where present. Protein extracts were prepared from different animals than used for RNA extraction (lines TG1 and TG2) or from one testis, whereas the other was used for RNA extraction (lines TG3 and TG4).



Determination of the Developmental Expression of the Transgenes during Spermatogenesis

The previously described results documented the organ specificity of transgene expression. However, those results could not establish that the transgene was expressing in the correct population of germinal cells, mid to late pachytene spermatocytes (Meistrich et al., 1985; Grimes et al., 1990; Kremer and Kistler, 1991). The time when germ cells in the immature mouse first reach particular stages of development is known (Bellvéet al., 1977). This makes it possible to assign initial transgene expression tentatively to a particular point in spermatogenesis by determining the earliest transgene expression in developing animals. RNA was extracted from testes of control mice and from those of the TG1-688 line at 7, 12, and 19 days after birth. A Northern blot was prepared and probed initially with the -141/+90 rat-specific probe A (Fig. 6A). This blot showed that the transgene was expressing even in 7-day-old mice, although the first pachytene spermatocytes are not normally seen in the mouse testis until animals are 14 days old (Bellvéet al., 1977). The blot was stripped and rehybridized to the +91/+804 nonspecific H1t probe B in order to show the onset of H1t expression in the control mouse testes (Fig. 6B). Although a more intense signal was detectable over the transgenic lanes, the control mouse showed no endogenous H1t expression in the 7- or 12-day-old samples, with the first expression occurring in the 19-day-old testes. This result suggested that the transgene in the TG1-688 line was expressing significantly earlier than the endogenous gene.


Figure 6: Developmental analysis of transgene expression by Northern blotting. A, RNA samples were prepared from the testes of animals at various times after birth, separated electrophoretically, blotted to nylon membranes, and hybridized with the P-labeled -141/+90 rat-specific probe A. B, after stripping, the blot was rehybridized to P-labeled +90/+804 nonselective H1t probe B.



Although the precise location of transgene expression might best be determined by in situ hybridization, an initial attempt to accomplish this with a S-riboprobe made from the -141/+90 rat H1t fragment was unsuccessful, perhaps because of the relatively short 90-bp hybridization target. Accordingly we decided to make use of an H1t-specific polyclonal antiserum that reacts with both mouse and rat H1t to test for the presence of H1t protein in early germ cells. With control adult mouse testis, this antiserum identified the nuclei of late pachytene spermatocytes, round spermatids, and early elongating spermatids, but no reaction was seen over the nuclei of early germ cells, elongated spermatids, or somatic cells of the testis (Fig. 7A). In striking contrast, testis sections of a mouse homozygous for the TG1-688 transgene showed an additional set of positively staining nuclei superimposed on those observed in the control mouse. Some tubules exhibited a distinctive pattern of staining of round nuclei in cells about the periphery of the tubule, which was never observed with material from nontransgenic animals (Fig. 7B). At higher magnification, it could be determined that early spermatogonia were generally unreactive with the antiserum (Fig. 7D) but that the earliest meiotic cells, preleptotene and leptotene spermatocytes, reacted very strongly (Fig. 7, E and F). Oddly, whereas these early spermatocytes were distinctively positive, spermatocytes beginning at about the zygotene phase became unreactive, and reactivity did not reappear until late pachytene spermatocytes. This accounts for the ring of unstained early pachytene nuclei seen in some tubules (Fig. 7, B and E). Precise identification of the earliest expressing cells was not possible with these sections, but they may be B type spermatogonia. Despite the unexpected detection of the transgene in early spermatocytes, expression was confined to germ cells, because no immune reaction was seen in nuclei outside the seminiferous tubules or within Sertoli cell nuclei.


Figure 7: H1t expression in testes of normal and transgenic animals detected by immunohistochemistry. Testes were fixed in Bouin's fluid, and routine paraffin-embedded sections were prepared. These were treated with a polyclonal antiserum raised to rat H1t, and the immune complexes were visualized by exposure to a peroxidase-conjugated second antibody and diaminobenzidine, as described under ``Experimental Procedures.'' The sections shown were not counterstained. A, low power view of nontransgenic mouse testis. The antiserum reacts with nuclei of mid to late pachytene spermatocytes (pc) as well as nuclei of round (rt) and elongating (et) spermatids. No reaction is seen over spermatogonia, early spermatocytes, elongated spermatids, or interstitial cells outside the tubules. B, low power view of transgenic testis from line TG1-688. Note that in contrast to A, nuclei are prominently labeled in some tubules around the periphery of the tubules. C, control slide in which the primary antiserum was omitted. D, E, and F are higher power views of sections from TG1-688. D, a tubule with unlabeled spermatogonia (sg), unlabeled early pachytene spermatocytes (pc), and heavily labeled round spermatids (rt). E, a tubule slightly more advanced than that of D, in which the spermatogonia have largely progressed to preleptotene spermatocytes (plc). The pachytene spermatocytes (pc) remain unlabeled, whereas round spermatids (rt) are heavily labeled. F, leptotene spermatocytes (lc) around the periphery of the tubules are labeled, whereas late pachytene spermatocytes (pc) are labeled as well as the elongating spermatids (et) found in tubules of this stage. Bar, 50 µm.



Although the antiserum does not distinguish between rat and mouse H1t, the most straightforward interpretation of the immunological results and the developmental Northern blot (Fig. 6) is that the transgene is expressed in this line significantly earlier in germ cell development than the endogenous gene. The loss of immunoreactivity in the zygotene and early pachytene cells is a mystery. It could reflect a temporary masking of antigenic determinants or destabilization of the association of H1t with chromatin and associated degradation of the protein. This same pattern of immunoreactivity was observed in the highly expressing TG4-4616 line, whereas the lower expressing TG3-4301 line had a pattern of immunoreactivity indistinguishable from the nontransgenic mouse testis (results not shown).

Testis-specific Expression of H1t-lacZ Fusion Gene

The previous results leave open the possibility that testis-directing sequences might lie within the H1t gene. To demonstrate that this is not the case, we isolated two founder lines that each contained an integrated fusion gene in which the -948/+71 fragment of H1t sequence was ligated to the E. coli lacZ structural gene. When a testis from the TG-LacZ-5371 line was lightly fixed and soaked in a histochemical stain solution for beta-galactosidase, it turned a dark blue within a matter of hours, indicating a positive reaction. Low power microscopic examination of the seminiferous tubules showed that staining was present in only a fraction of the cells, whereas no staining was observed in tubules of a nontransgenic control animal (Fig. 8). The second founder line did not give a positive histochemical stain for beta-galactosidase in the testes or in any other organ tested. To quantitate beta-galactosidase activity in the TG-LacZ-5371 line, extracts were prepared from a number of organs. Only the testes showed an activity significantly above (6-8-fold) the background levels seen in nontransgenic controls (Fig. 9).


Figure 8: Expression of lacZ transgene in seminiferous tubule whole mounts. Teased testis tubules from a mouse of the TG-LacZ-5371 line (A) or from a nontransgenic control mouse (B) were stained for 2 days to detect beta-galactosidase activity. Intact tubules were photographed using a deep red filter to enhance the contrast of the blue reaction product. No blue color was seen in the control tubules, and gray spots visible on the photograph are due to elongated spermatid nuclei. Bar, 100 µm.




Figure 9: beta-Galactosidase activity in homogenates of various organs. Representative organs were assayed for beta-galactosidase activity as described under ``Experimental Procedures.''



To determine the site of transgene expression within the testes of the 5371 line, histological cross sections were prepared. Staining was observed over pachytene spermatocytes and round spermatids but not over other cell populations in the tubules (Fig. 10), mimicking the location of H1t itself (compare Fig. 7). It is puzzling that the staining of spermatocytes and round spermatids was quite variable, with some cells staining darkly and others in the same cross section remaining virtually unstained (Fig. 10). This pattern was observed in each of three animals examined from this line.


Figure 10: Expression of lacZ transgene in seminiferous tubule cross sections. Teased testis tubules from a mouse of the TG-LacZ-5371 line (A and C) or from a nontransgenic control mouse (B and D) were stained for 2 days to detect beta-galactosidase activity. Tubules were then embedded for light microscopy, sectioned, and counterstained with nuclear fast red. The same cross section was photographed through a light green filter to identify nuclei (A and B) or through a deep red filter to enhance the contrast of the indigo reaction product (C and D). beta-Galactosidase activity was observed over round spermatids (thin arrow) and pachytene spermatocytes (thick arrow) but not over early germ cells found in the outermost germ cell layer of the tubules or over elongated spermatids found near the lumen. Bar, 30 µm.




DISCUSSION

Sequences Regulating Rat H1t Expression Lie within a Discrete Chromosomal Region

H1t is a tissue-specific member of the H1 gene family. Its expression is limited to late pachytene spermatocytes. In this study we have defined the DNA region that conveys this tissue specificity by introducing both the natural rat H1t coding sequence as well as an H1t-lacZ fusion gene into transgenic mice. Genomic fragments with 2.4 kb of upstream sequence and 4 kb of downstream sequence resulted in high level, testis-specific expression of the natural rat H1t transgene. Fragments truncated to within 141 bp upstream or 837 bp downstream of the 741-bp gene maintained testis-specific expression, although with variable levels of expression. An H1t-lacZ fusion gene with 0.95 kb of 5`-flanking sequence also expressed specifically in the correct germ cell populations. Thus the sequences sufficient to activate expression in the male germ line yet prevent expression in somatic cells must lie within the 1019-bp fragment running from -952 to +71. Correct expression may indeed be driven by merely the -141/+71 fragment, although it is also possible that redundant control sequences are found in the -952 to -141 and downstream regions so that correct expression is compatible with deletion of either region individually but not in combination.

Whereas all of the genomic fragments yielded testis-specific expression, the levels of expression in the most 5`- and 3`-truncated fragments (TG3 and TG4) were generally not so high as with the full-length fragment (TG1). An earlier study in somatic cells transfected with the H1t 5`-flanking region fused to the chloramphenicol acetyltransferase gene indicated that stimulatory sequences lie between -368 and -693 (Kremer and Kistler, 1992). The lower expression of two of the TG3 lines (TG3-5592 and TG3-5597) compared with the TG1 and TG2 lines agrees with this conclusion, although expression from the remaining TG3 line (TG3-4301) was stronger and comparable with the TG2 lines. The most variable levels of expression were found with TG4 lines, which deleted downstream sequences past +1578. The H4t gene lies within the region deleted (Fig. 1), and it is plausible to suppose that sequences neighboring H4t could have a stimulatory effect on both the H1t and H4t genes.

Although the H4t gene is expressed in late pachytene spermatocytes, it is also expressed in the brain and to a lesser extent in liver (Wolfe et al., 1989). Accordingly, in the case of H4t, expression in spermatocytes is not coupled to complete repression in somatic organs. Because the H4t gene encodes a protein with the same amino acid sequence as somatic H4 (Grimes et al., 1987), its expression would not have an obvious functional consequence in somatic cells. Thus, controls on its somatic expression may not be as stringent as for the other germ cell-specific histone variants.

Sequences Responsible for Testis-specific Expression Remain Speculative

Most H1 genes code for members of the standard somatic H1 family (H1a-e), and analysis of their transcriptional control has focused on cell cycle regulation in cultured cells (reviewed in Heintz(1991)) rather than expression in tissues. These studies have identified a set of four conserved sequences lying within 100 bp upstream of the transcriptional initiation site of all standard H1 genes (Coles and Wells, 1985). Two of these are binding sites for novel, H1-specific factors, the H1 box (Dalton and Wells, 1988) and the CCAAT region (van Wijnen et al., 1988; Gallinari et al., 1989; Martinelli et al., 1994). The remaining two DNA elements (GC box and TATA box) are presumed to interact with common transcription factors. As H1t has each of these conserved promoter elements, they do not seem to account for its tissue specificity. Recently Grimes and colleagues (Grimes et al., 1992a, 1992b) have identified a binding activity in testis nuclear extracts for a palindrome found between the GC and CCAAT boxes in the promoter region of the H1t genes of several species but absent in standard somatic H1 promoters. Whether this factor plays a role in H1t expression remains to be established. In this context it is interesting to note that Chae and Lim(1992) have reported a possible repressor of the testis-specific H2B gene in extracts of immature rat testes that bound to a site just 3` of the TATA box.

The possible role of distant sequences on the expression of standard H1 genes is not well explored. However, participation of distant upstream sequences is documented for expression of the mouse H1^o gene. H1^o is a minor linker histone whose expression is associated with the onset of a nonproliferative, differentiated phenotype. Unlike a standard H1 gene, the H1^o gene encodes a polyadenylated mRNA. Although its promoter shares three of the conserved elements of the standard H1 genes, an apparent histone H4 promoter element is substituted for the usual H1-specific CCAAT sequence (Breuer et al., 1989, 1993; Khochbin and Lawrence, 1994). Studied by transfection in mouse F9 embryonal carcinoma cells, basal expression of the mouse H1^o 5`-flanking region was shown to depend on an 80-bp region at about -500 that bound several nuclear proteins (Breuer et al., 1993). The H1^o gene is induced by vitamin A in F9 cells, and this induction is associated with a pair of tandem retinoic acid response elements located just 3` to this 80-bp region necessary for basal expression (Breuer et al., 1989, 1993). Although the relevance of this extreme H1 variant to H1t is uncertain, it provides a precedent for influence of relatively distant sequences on mammalian H1 gene expression. Roles for sequences upstream of the proximal promoter region have also been proposed through extensive study of a cell cycle-regulated human H4 gene (reviewed in Stein et al.(1992)).

Why Is the H1t Gene Expressed Prematurely in Some Transgenic Lines

The natural H1t gene is expressed only in mid to late pachytene primary spermatocytes beginning about stage VII in the rat as determined by appearance of the protein in young animals (Seyedin and Kistler, 1980), by analysis of H1t synthesis in purified germ cell populations (Meistrich et al., 1985), and by detection of H1t mRNA via in situ hybridization (Kremer and Kistler, 1991). The results presented here for the developmental onset of H1t mRNA expression in normal mouse testis as well as the immunohistological detection of H1t protein confirm this mid/late pachytene expression. It was therefore surprising to find that two of three transgenic lines examined expressed the transgene prematurely, clearly in preleptotene spermatocytes, and perhaps even in B type spermatogonia. Although we have not checked for subtle effects, the early expression of rat H1t in these mice had no obvious effects on their fertility.

Several explanations for the early expression of the transgene can be considered. First, it is possible that an additional cis-acting DNA locus that lies outside the 6.8-kb region we have investigated is required to fine tune H1t appearance. This seems unlikely because one of the the three lines did not show premature transgene expression. A second possibility is that the natural gene also becomes active in late spermatogonia/early spermatocytes but at too low a level to be detected by the techniques applied. The multicopy transgene might simply yield a detectable signal coincident with the earliest activation of the gene through a gene dosage effect. A third explanation is that having many copies of the gene, all integrated in tandom (as is the general case for transgenes (Jaenisch, 1988) but has not been investigated in the lines under discussion), leads to an enhanced probability of expression when necessary factors are limiting, perhaps because the effect of the many tandem repeats is to build a higher local concentration of the appropriate factors than would otherwise occur. Although this might lead to a nonproductive distribution of factors over different promoters, it might also lead eventually to a productive clustering on one or more of the tandom repeats. A fourth explanation is that a negative-acting factor in limiting concentration is titrated out by multiple transgene copies. At the present time we do not have any reason to support one of these possibilities more strongly than the others. It is curious, however, that the various testis-specific histone variants appear at slightly different developmental times, TH3 in spermatogonia, TH2A and TH2B in early spermatocytes, and H1t and H4t in late spermatocytes (Meistrich et al., 1985; Kim et al., 1987; Kremer and Kistler, 1991; Grimes et al., 1987). It is unknown whether any of the factors that determine male germ cell-specific expression are shared among these genes, but if so, there could be some sort of titration of DNA response elements during germ cell development, and the presence of multicopies may somehow change the outcome of this titration. It may be relevant that a somewhat comparable situation has been described for the transgenic expression of the human keratin-1 gene, which is normally expressed only in the suprabasal layers of the skin. In transgenic mice, expression of human keratin-1 was restricted to the skin, but in contrast to the natural gene, the transgene was expressed prematurely in the mitotically dividing basal cells (Rosenthal et al., 1991).

Competition between Transgene and Endogenous H1t Genes

In some transgenic lines, e.g. TG1-688 and TG2-97, levels of the endogenous mouse H1t protein were markedly reduced. This result could be explained by competition among excess protein molecules attempting to bind to limited places available on chromatin. In a recent study, Brown and Sittman(1993) observed that overproduction of H1e or H1c in somatic cell lines led to correspondingly reduced levels of other H1 variants, although the mechanism for the reduction of the endogenous H1 variants was not experimentally addressed. Competition for H1(t)-specific transcription factors could also account for a reduced level of mouse H1t protein if the mouse H1t mRNA was also low. Some lines of transgenic mice expressing a p53 promoter-chloramphenicol acetyltransferase fusion, which was expressed at high levels in the testes, displayed a reduced level of p53 mRNA in the testes, associated with a failure of cells to complete meiosis (Rotter et al., 1993). One explanation suggested for this effect was a competition of the transgene and endogenous p53 promoters for limiting transcription factors.

In summary, results presented here indicate that the sequence elements conferring spermatocyte-specific expression to H1t are restricted to at most 1 kb of 5`-flanking sequence and may lie within as little as 212 bp. We are thus encouraged to look to this region for the DNA sequences responsible and their associated binding factors.


FOOTNOTES

*
This work was supported by Grant HD10793 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: US EPA Environmental Research Laboratory, 1 Sabine Island Dr., Gulf Breeze, FL 32561.

Present address: Laboratoire de Génétique des Virus Oncogénes, 39 rue Camille Desmoulins PR2 3C2, Villejuif Cedex 94805, France.

**
To whom correspondence should be addressed: Dept. of Chemistry, 730 S. Main St., Columbia, SC 29208. Tel.: 803-777-4799; Fax: 803-777-9521.

(^1)
The abbreviations used are: bp, base pair(s); kb, kilobase(s); TBS, Tris-buffered saline; TBST, Tris-buffered saline with Tween; UTR, untranslated region; HMG, high mobility group.

(^2)
R. J. Oko, V. Jando, C. L. Wagner, W. S. Kistler, and L. S. Hermo, submitted for publication.


ACKNOWLEDGEMENTS

We thank Linda Bade for help with preparation of histological sections, Clark Millette for assistance in identifying H1t immunoreactive germ cells in transgenic animals, and Richard Showman for the use of microscopic facilities. We are grateful to Richard Palmiter for providing placI.

Note Added in Proof-Recently vanWert et al.(1995) described testis-specific expression of rat H1t in transgenic mice carrying a genomic fragment identical to TG1 (Fig. 1).


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