(Received for publication, September 1, 1995; and in revised form, October 31, 1995)
From the
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.
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 ()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.
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.
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
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.
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).
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).
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).
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
-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:
-Galactosidase activity in
homogenates of various organs. Representative organs were assayed for
-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 -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).
-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.
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.
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 gene. H1
is a minor linker histone whose expression is associated with the
onset of a nonproliferative, differentiated phenotype. Unlike a
standard H1 gene, the H1
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
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
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)).
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).
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.
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).