Biomedical subjects

W S Kistler

Publications and source records attributed to W S Kistler.

At least 19 recordsLinked to original sources

PubMed2001

Mice with a targeted disruption of the H1t gene are fertile and undergo normal changes in structural chromosomal proteins during spermiogenesis.

H1t is an H1 histone variant unique to late spermatocytes and early spermatids. Using gene targeting and embryonic stem cell technologies, we have produced mice with a disrupted H1t gene. Homozygous H1t-null mice have normal fertility and show no obvious phenotypic consequence due to the lack of this histone. Biochemical and immunohistochemical approaches were used to show that normal changes in chromosomal proteins occurred during spermatid development, including the appearance and disappearance of transition proteins 1 and 2. Both protamines 1 and 2 are present in normal amounts in sonication-resistant spermatid nuclei from H1t-null mice. Analysis of H1 histones by quantitative gel electrophoresis in enriched populations of pachytene spermatocytes and round spermatids showed that the lack of H1t is only partially compensated for by somatic H1s, so that the chromatin of these cells is H1 deficient. Because H1t is thought to create a less tightly compacted chromatin environment, it may be that H1-deficient chromatin is functionally similar to chromatin with H1t present, at least with respect to permitting spermatogenesis to proceed.

Alleles↗

PubMed2001

Characterization of the H1t promoter: role of conserved histone 1 AC and TG elements and dominance of the cap-proximal silencer.

H1t is a testis-specific variant histone 1 gene transcribed in pachytene spermatocytes. As part of a program to understand its transcriptional control, we have investigated the effect of the cap-proximal, GC-rich silencer element in the context of various lengths of upstream sequence. By transient transfection of NIH 3T3 cells, we showed that a targeted mutation in the silencer has a large (>10-fold) effect on reporter gene expression, regardless of the length of upstream sequence present. No other discrete silencing activity was observed in the upstream region extending to nucleotide -1842. Similarly, when the silencer mutation was introduced into the natural gene, H1t expression was readily detected in permanently transfected cells by both RNase protection and Western blot analysis, regardless of the extent of 5' or 3' flanking genomic DNA. In constructs with the mutated silencer, we showed interdependence of the characteristic H1 AC and TG box regulatory elements. Promoter up-regulation occurred only when both were intact, and possibly identical binding factors were demonstrated for each by electrophoretic mobility shift assays. In view of its precisely regulated but limited expression, it is interesting that H1t retains all the promoter elements known to activate standard H1 genes, including the TG/AC unit, SP1 site, and CCAAT element. Their presence emphasizes the apparent dominance of the silencer element in most cells.

3T3 Cells↗

PubMed2000

Elimination of male germ cells in transgenic mice by the diphtheria toxin A chain gene directed by the histone H1t promoter.

Expression of the diphtheria toxin A-chain gene was directed to the male germ line by fusion to 1 kilobase of the 5'-flanking DNA of the rat histone H1t gene. Two independent lines of mice were established that expressed the toxic transgene. Female carriers were fertile; males were sterile although otherwise apparently normal. Adult transgenic males had very small testes that were virtually devoid of germ cells. A developmental study showed that germ cells survived until late fetal life but that testes of 3-day-old transgenic mice were severely depleted of prospermatogonia. During postnatal development of transgenic animals, remaining germ cells progressed to the pachytene stage of meiosis in 10% to 30% of tubular cross sections but degenerated before the completion of meiosis. By 3 mo of age the residual germ cells had almost completely disappeared. These transgenic lines demonstrate the complete tissue specificity of the H1t promoter and reveal a period of its activity just prior to formation of the definitive adult spermatogonial stem cell population. Whereas full expression of H1t occurs only in mid to late pachytene spermatocytes, one or more of the factors that impart tissue specificity to its expression must be transiently activated in the neonatal germ line. This report discusses the possibility that this genetic technique for eliminating germ cells may have practical application in making recipients for spermatogonial stem cell transplantation.

Animals↗

PubMed1997

The testis-specific histone H1t gene is strongly repressed by a G/C-rich region just downstream of the TATA Box.

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.

3T3 Cells↗

PubMed1997

Characterization of the promoter region of the rat testis-specific histone H1t gene.

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.

Animals↗

PubMed1996

Sequential expression of nucleoproteins during rat spermiogenesis.

Transition proteins and protamines are highly basic sperm-specific nuclear proteins that serve to compact the DNA during late spermiogenesis. To understand their sequential role in this function, transition protein 1 (TP1), transition protein 2 (TP2), and protamine 1 (P1) were assayed by polyacrylamide gel electrophoresis in pools of microdissected, staged seminiferous tubule segments in the rat. The results were compared with immunocytochemical analyses of squash preparations from accurately identified stages of the epithelial cycle. TP2 was the first to appear as a faint band at stages IX-XI, followed by high levels at stages XII-XIV of the cycle. TP1 showed a low expression at stage XII of the cycle and peaked at stages XIII-I, whereas protamine 1 first appeared at stage I of the cycle and remained high throughout the rest of spermiogenesis. Immunocytochemical analyses and Western blots largely confirmed these results: TP2 in steps 9-14, TP1 in steps 12-15, and P1 from late step 11 to step 19 of spermiogenesis. We propose that TP2 is the first nucleoprotein that replaces histones from the spermatid nucleus, and its appearance is associated with the onset of nuclear elongation. TP1 shows up along with the compaction of the chromatin. The two transition proteins seem to have distinct roles during transformation of the nuclei and compaction of spermatid DNA.

Animals↗

PubMed1996

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.

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.

Animals↗

PubMed1996

Chromatin reorganization in rat spermatids during the disappearance of testis-specific histone, H1t, and the appearance of transition proteins TP1 and TP2.

Transition proteins replace testis-specific histones and are finally replaced by protamines in the nucleus of germ cells during spermiogenesis. In this study, immunoperoxidase and immunogold localization were used to determine both qualitatively and quantitatively the intracellular distribution of testis-specific histone (H1t), transition protein 1(TP1), and transition protein 2 (TP2) during rat spermatogenesis. H1t labeling was concentrated over heterochromatin in the nucleus of late-pachytene spermatocytes and spermatids up to mid-steps 10. In step 9 spermatids, H1t was confined to the caudal end of the nucleus where heterochromatin was still present, while in early step 10 spermatids, only a few of the nuclei remained caudally labeled. In late step 10 spermatids, a fibrillar chromatin network was distributed throughout the nucleus coincident with the loss of H1t. A statistically significant rise in TP1 and TP2 labeling density over control values was first encountered in the nucleus of step 11 spermatids coincident with the initiation of condensation of the fibrillar chromatin. The TP1 and TP2 labeling density progressively increased in nucleus of step 11-13 spermatids with the apical to caudal condensation of the fibrillar chromatin, In step 13 spermatids, the chromatin was homogeneously condensed throughout the nucleus. In the case of TP1, the nuclear labeling density gradually declined after step 13 and disappeared by step 17. In the case of TP2, the nuclear labeling density disappeared by step 16. This study shows that, coincident with the loss of H1t, the chromatin of the spermatid is reorganized into a fibrillar network, whereas, coincident with the appearance and progressive increase of TP1 and TP2, the fibrillar chromatin condenses in an apical to caudal direction in the nucleus of the spermatid. Thus the remodeling of chromatin structure during spermiogenesis appears to be a two-step process that is sequentially influenced by the loss of spermatid-specific histones and the appearance of transition proteins.

Animals↗

PubMed1995

Ultrastructural immunolocalisation of histones (H2B, H3, H4), transition protein (TP1) and protamine in rabbit spermatids and spermatozoa nuclei. Relation to condensation of the chromatin.

The histones H2B, H3 and H4, the transition protein TP1 and protamine were localised using ultrastructural immunocytochemistry in nuclei of rabbit spermatids and spermatozoa. Histones are present in round spermatid nuclei and are lost during the elongation of nuclei. TP1 and protamine appear simultaneously in all nuclei during this period. TP1 is located at the periphery of chromatin cords, while protamine seems to be located at random in the same cords. TP1 is lost in most elongated spermatids during step 13 of spermiogenesis, and the protamine stays in all sperm nuclei. TP1 remains present in some old spermatids and ejaculated spermatozoa. In the rabbit, 3-6% of sperm nuclei decondense spontaneously. Most are characterised by a retention of TP1. Respective roles of TP1 and the protamine in spermatid nuclear condensation are discussed.

Animals↗

PubMed1994

Identification of a functional cyclic adenosine 3',5'-monophosphate response element in the 5'-flanking region of the gene for transition protein 1 (TP1), a basic chromosomal protein of mammalian spermatids.

Transition protein 1 (TP1) is a small basic chromosomal protein that appears in mammalian spermatids during the period of chromatin condensation. The gene for TP1 from several species contains an apparent cAMP response element (CRE) in the immediate 5'-flanking region. The recent identification of high expression of the novel CRE-activating protein (CREM tau) in advanced testicular germ cells provided a stimulus to ask whether or not the TP1 CRE is functional. To this end we show both by gel retardation and by footprint assays that TP1 CRE forms specific bound complexes with proteins in whole testis nuclear extracts and that these complexes involve CREM as evidenced by recognition by a specific antibody. In addition, the TP1 CRE forms specific bound complexes with bacterially expressed CREM tau. Finally, the TPI CRE conveys protein kinase A-dependent induction to a linked chloramphenicol acetyl transferase gene when transfected into JEG-3 cells. Accordingly, TP1 is a good candidate for a testis-specific gene subject to CREM tau regulation.

Animals↗

PubMed1993

Immunohistochemical localization of spermatid nuclear transition protein 2 in the testes of rats and mice.

Transition protein 2 (TP2) of the rat was isolated by differential precipitation with trichloroacetic acid, chromatography over Bio-Rex 70, and preparative gel electrophoresis. A polyclonal rabbit antiserum was raised that did not cross-react with unrelated acid-soluble proteins from liver or testes. The antiserum was used to identify TP2-related proteins obtained from testes of mice, hamsters, guinea pigs, rabbits, and boars by Western blotting. Immunohistochemical techniques were used to localize TP2 in paraffin-embedded testis sections from mice and rats. In both species, TP2 was first detected in spermatids that had essentially completed the morphological change from a round to an elongate nucleus and that were undergoing chromosomal condensation (spermatids of step 13 in rat and step 12 in mouse). TP2 was retained in spermatid nuclei until early step 16 in the rat and step 14 in the mouse. Serial sections of rat testis exposed separately to antisera to TP1 and TP2 showed that the great majority of labeled tubules were reactive to both antisera. However, in occasional tubules, TP1 reactivity was retained in relatively late spermatids that were negative for TP2. Thus both TP1 and TP2 appear in the nucleus essentially simultaneously, in association with the beginning of chromatin condensation and at a point well after much of the nuclear shaping has occurred.

Animals↗

PubMed1992

Analysis of the promoter for the gene encoding the testis-specific histone H1t in a somatic cell line: evidence for cell-cycle regulation and modulation by distant upstream sequences.

Gene H1t encodes a testis-specific variant of the H1 histone family expressed in pachytene spermatocytes during the meiotic phase of spermatogenesis. Fusions between various upstream fragments of the H1t gene and the chloramphenicol acetyltransferase-encoding reporter gene were analyzed in mouse L cells by both transient and permanent transfection. Expression of the minimal promoter [174 nucleotides (nt) upstream from the transcription start point] was enhanced by sequences extending to nt -693, but was reduced in constructs with kb of upstream sequence. Using synchronized cells, expression was at least twofold higher in growing than in inhibited cells. Thus, the H1t promoter is modulated both positively and negatively by distant upstream sequences, and it displays some of the S-phase-dependent character of a replication-dependent histone.

Animals↗

PubMed1991

Localization of mRNA for testis-specific histone H1t by in situ hybridization.

H1t is a testis-specific H1 histone variant that appears in germ cells during the meiotic prophase of mammalian spermatogenesis. Using a tritiated antisense RNA probe, H1t mRNA was identified by in situ hybridization in the mid and late pachytene spermatocytes found in seminiferous tubules of approximately stages VII to XIII.

Animals↗

PubMed1990

Isolation of a genomic clone encoding the rat histone variant, H1d.

Mammals contain a family of five closely related H1 histone variants (H1a-e) as well as two less closely related forms, H10 and H1t. We have sequenced a rat genomic clone that encodes one of the standard H1 variants. An RNA transcript of the gene was made with bacteriophage SP6 RNA polymerase and translated in a cell-free system. The protein synthesized in vitro was identified as variant H1d by its electrophoretic mobility.

Amino Acid Sequence↗

PubMed1989

Nucleotide sequence of the Stp-1 gene coding for rat spermatid nuclear transition protein 1 (TP1): homology with protamine P1 and assignment of the mouse Stp-1 gene to chromosome 1.

Spermatid transition protein 1 (TP1) is a 54 amino acid (aa), highly basic chromosomal protein found in mammals during the brief period when histones are being replaced by protamines in the haploid phase of spermatogenesis. Using a cDNA clone as probe, we have isolated the gene (Stp-1) coding for rat TP1 from a population of recombinant bacteriophage lambda. The nucleotide (nt) sequence was established from a point 126 nt upstream from the mRNA cap site to a point about 30 nt downstream from the predicted site of polyadenylation. The gene contains a single intron separating the codon for aa 45 of the mature protein. Comparison of the nucleotide sequences for Stp-1 and the mouse gene coding for protamine P1 suggests a possible evolutionary relationship. Southern blot hybridization to genomic DNA isolated from a panel of mouse-hamster somatic cell hybrids unambiguously mapped Stp-1 to mouse chromosome 1.

Amino Acid Sequence↗

PubMed1988

A cytochemical study of the transcriptional and translational regulation of nuclear transition protein 1 (TP1), a major chromosomal protein of mammalian spermatids.

Immunocytochemical localization and in situ hybridization techniques were used to investigate the presence of spermatid nuclear transition protein 1 (TP1) and its mRNA during the various stages of spermatogenesis in the rat. A specific antiserum to TP1 was raised in a rabbit and used to show that TP1 is immunologically crossreactive among many mammals including humans. During spermatogenesis the protein appears in spermatids as they progress from step 12 to step 13, a period in which nuclear condensation is underway. The protein is lost during step 15. An asymmetric RNA probe generated from a TP1 cDNA clone identified TP1 mRNA in late round spermatids beginning in step 7. The message could no longer be detected in spermatids of step 15 or beyond. Thus, TP1 mRNA first appears well after meiosis in haploid cells but is not translated effectively for the several days required for these cells to progress to the stage of chromatin condensation. Message and then protein disappear as the spermatids enter step 15. In agreement with a companion biochemical study (Heidaran, M.A., and W.S. Kistler. J. Biol. Chem. 1987. 262:13309-13315), these results establish that translational control is involved in synthesis of this major spermatid nuclear protein. In addition, they suggest that TP1 plays a role in the completion but not the initiation of chromatin condensation in elongated spermatids.

Animals↗

PubMed1987

Transcriptional and translational control of the message for transition protein 1, a major chromosomal protein of mammalian spermatids.

The spermatid transition proteins comprise a set of basic chromosomal proteins that appear during the period when spermatids are undergoing nuclear elongation and condensation, about midway between the end of meiosis and the release of spermatozoa from the seminiferous tubule. The transition proteins replace the histones but are themselves subsequently replaced by protamines, and they are not found in sperm nuclei. We have used a cDNA clone for the smallest transition protein (TP1, 54 amino acids) to show that its message first appears postmeiotically in late round spermatids. Thus production of TP1 is an example of haploid gene expression. The message remains translationally inactive for some 3-4 days before translation occurs in early elongating spermatids. While translationally repressed, TP1 message is nonpolysomal and has a discrete size of about 590 bases, including a 140 residue poly(A) tail. In contrast, polysome-associated message is of heterogeneous size due to variability of poly(A) lengths.

Animals↗

PubMed1987

Nuclear transition protein 2 (TP2) of mammalian spermatids has a very basic carboxyl terminal domain.

Nuclear transition protein 2 (TP2) along with TP1 are major basic chromosomal proteins of rat spermatids during the period of transition from histone-associated to protamine-associated DNA. TP2 isolated by reversed phase high pressure liquid chromatography was cleaved with S. aureus V8 protease to yield two fragments. The complete amino acid sequence of the 27 residue peptide assigned to the carboxyl terminus was established. It contains most of the basic residues of the protein and is likely to be a major site of DNA binding. Thus, TP2 is differentiated from core histones in having its basic domain at the carboxyl rather than amino terminal end.

Amino Acid Sequence↗