Testis-specific transcriptional control.

In the testis, tissue-specific transcription is essential for proper expression of the genes that are required for the reproduction of the organism. Many testis-specific genes are required for mitotic proliferation of spermatogonia, spermatocytes undergoing genetic recombination and meiotic divisions, and differentiation of haploid spermatids. In this article we describe some of the genes that are transcribed in male germinal cells and in non-germinal testis cells. Because significant progress has been made in examination of promoter elements and their cognate transcription factors that are involved in controlling transcription of the testis-specific linker histone H1t gene in primary spermatocytes, this work will be reviewed in greater detail. The gene is transcriptionally active in spermatocytes and repressed in all other germinal and non-germinal cell types and, therefore, it serves as a model for study of regulatory mechanisms involved in testis-specific transcription.

Transcriptional control of the testis-specific histone H1t gene.

The mammalian testis-specific linker histone H1t is synthesized only in pachytene primary spermatocytes during spermatogenesis. In this review we summarize some of the progress made in our laboratory and in other laboratories in understanding transcriptional regulation of this gene. The gene is transcriptionally active in pachytene primary spermatocytes and is repressed in all other germinal and non-germinal cell types. To place the transcriptional control of the testis-specific histone H1t gene in the proper context, we briefly review recent literature concerning mammalian linker histone genes in general and we compare and contrast these with the testis-specific histone H1t gene.

Microsoft Word macro for analysis of cytosine methylation by the bisulfite deamination reaction.

Cytosine methylation at CpG dinucleotides is an important control mechanism in development, differentiation, and neoplasia. Bisulfite genomic sequencing and its modifications have been developed to examine methylation at these CpG dinucleotides. To use these methods, one has to (i) manually convert the sequence to that produced by bisulfite conversion and PCR amplification, taking into account that cytosine residues at CpG dinucleotides may or may not be converted depending on their methylation status, (ii) identify relevant restriction sites that may be used for methylation analysis, and (iii) conduct similar steps with the other DNA strand since the two strands of DNA are no longer complementary after bisulfite conversion. To automate these steps, we have developed a macro that can be used with Microsoft Word. This macro (i) converts genomic sequence to modified sequence that would result after bisulfite treatment facilitating primer design for bisulfite genomic sequencing and methylation-sensitive PCR assay and (ii) identifies restriction sites that are preserved in bisulfite-converted and PCR-amplified product only if cytosine residues at relevant CpG dinucleotides are methylated (and thereby not converted to uracil) in the genomic DNA.

Testis-specific histone H1t gene is hypermethylated in nongerminal cells in the mouse.

The testis-specific histone H1t gene is expressed only in pachytene primary spermatocytes during spermatogenesis. There is a correlation between the specific binding of testis nuclear proteins to a rat histone H1t promoter sequence, designated the H1t/TE element, and the onset of transcription in primary spermatocytes. Our laboratory has shown that mice bearing the rat gene with a deletion of the TE promoter element and replacement with a heterologous stuffer DNA fragment fail to express the rat H1t transgene in any tissue. In this study we report that five CpGs located within the H1t proximal promoter, including two CpGs located within the essential TE promoter element, contain unmethylated cytosines in vivo in genomic DNA derived from primary spermatocytes where the H1t gene is expressed. All seven CpGs are hypermethylated in vivo in genomic DNA derived from liver cells where gene expression is repressed. Further, in vitro methylation of an H1t promoter-driven reporter plasmid markedly reduced expression in a transient transfection assay system. These results suggest that cytosine methylation may contribute to the transcriptional silencing of the testis-specific histone H1t gene in nonexpressing tissues such as liver.

Binding of nuclear proteins to an upstream element involved in transcriptional regulation of the testis-specific histone H1t gene.

The testis-specific histone H1t is synthesized during spermatogenesis exclusively in late pachytene primary spermatocytes. Transcription of the H1t gene is repressed in every tissue except testis. Within the testis, transcription is repressed during development before the spermatocyte stage and in later stages of germinal cell maturation. Mechanisms involved in transcriptional repression of the H1t gene are unknown. To assess the contribution of upstream H1t promoter sequence to transcriptional silencing in nonexpressing cells, H1t-promoted reporter vectors were constructed using pGL3 Basic. Transient expression assays with these reporter vectors driven by H1t promoter deletions allowed us to identify a region from 948 to 780 bp upstream from the H1t transcriptional initiation site that functions as a silencer. Examination of nuclear protein binding to this DNA regulatory region by electrophoretic mobility shift assays using extracts from C127I cells, rat testis, and pachytene spermatocytes revealed a low mobility band produced only by nuclear proteins derived from nonexpressing cells that may contain proteins that repress H1t gene transcription.

Localization of upstream elements involved in transcriptional regulation of the rat testis-specific histone H1t gene in somatic cells.

The testis-specific histone H1t is synthesized exclusively in late pachytene primary spermatocytes during spermatogenesis. The mechanisms involved in transcriptional repression of the H1t gene during development before the spermatocyte stage and in later stages of germinal cell maturation and in nonexpressing somatic tissues are unknown. To assess the contribution of the upstream DNA sequence to H1t transcriptional silencing in nonexpressing cells, a set of histone H1t-promoted reporter vectors was constructed. Transient transfection of mouse C127I cells with these reporter vectors allowed us to identify a transcriptional silencer located between 948 base pairs (bp) and 780 bp upstream from the H1t transcriptional initiation site. Histone H1t-promoted luciferase activity increased 4-fold when the region between 948 bp and 875 bp upstream from the transcriptional initiation site was eliminated. Addition of a 73-bp rat H1t promoter fragment (-948 to -875, containing the 5' portion of the silencer region) to a site immediately upstream from the histone H1d proximal promoter led to significantly reduced luciferase expression upon transient transfection (56% in C127I cells and 44% in HeLa cells). Nuclear proteins were found to bind to DNA within the H1t silencer region when assayed by in vitro deoxyribonuclease (DNase) I footprinting. Thus, our data suggest that an active transcriptional silencer mechanism involving a specific and autonomous H1t promoter element (nucleotides -948/-875) may be operative to minimize expression of the H1t gene in nontesticular cells.

Cloning and characterization of the prostate-specific membrane antigen promoter.

Prostate-specific membrane antigen (PSMA) is a protein that is expressed predominantly in normal prostate epithelial cells and in most adenocarcinomas of the prostate (Cap) and in virtually all Cap metastases. In this article we describe the cloning of a 2-kb human genomic DNA fragment containing the 5' upstream untranslated region of the PSMA gene and present evidence that it provides promoter activity. When the DNA fragment was cloned into transient expression vectors to examine promoter activity, the vectors were functional in promoting expression in several prostate and nonprostate cell lines in transient transfection assays. A 614-bp fragment derived from the 3' end of the 2-kb fragment may represent the minimal PSMA promoter as determined by deletion mutagenesis. The 2-kb fragment compared with the 614-bp fragment provided higher expression levels when using prostate-derived cell lines (DU 145 and LNCaP). The increased transcription using the 2-kb fragment was not as great in non-prostate cell lines. Little or no transcription over basal levels was seen with a 232-bp promoter fragment. When the concentration of dihydrotestosterone was depleted or supplemented in the growth medium, no significant effect was seen on PSMA-promoted transient expression in LNCaP cells, a prostate cell line. J. Cell. Biochem. 74:395-405, 1999. Published 1999 Wiley-Liss, Inc.

The TE promoter element of the histone H1t gene is essential for transcription in transgenic mouse primary spermatocytes.

Transcriptional activation of the testis-specific histone H1t gene occurs in pachytene primary spermatocytes during spermatogenesis. Specific binding of testis nuclear proteins to a rat histone H1t promoter sequence, designated the H1t/TE element, correlates with the onset of transcription. This element, located between the H1t/AC box and the H1t/CCAAT box, contains inverted repeats of a shorter element. When the native rat H1t gene along with flanking sequences, including 2453 base pairs (bp) upstream and 3784 bp downstream from the coding region, was microinjected into mouse embryos, the offspring of the resulting transgenic mice transcribed the transgene in a tissue-specific manner and only in primary spermatocytes. In the present study the TE promoter element was deleted and replaced with a heterologous stuffer DNA fragment. When the mutant rat DNA fragment was used to create transgenic mice, offspring of the mice bearing the promoter mutation did not transcribe the rat H1t gene in any tissue. On the other hand, transcription of the rat H4t transgene, which is located approximately 1.5 kilobases downstream from the H1t gene, occurred in these animals. Therefore, these studies support the hypothesis that the TE element is essential for enhanced testis-specific transcription of the H1t gene in primary spermatocytes.

Regulation of transcription of the testis-specific histone H1t gene by multiple promoter elements.

This is a review of mechanisms that contribute to testis-specific transcription of the histone H1t gene. The mammalian testis-specific histone H1t gene is transcribed only in primary spermatocytes during spermatogenesis. Linker histones bind to DNA and contribute to chromatin condensation by formation of the 30 nm chromatin fiber. Furthermore, linker histones contribute to regulation of transcription of specific genes. Histone H1t, which binds more weakly to DNA than the other six known linker histones, is expressed in cells that are involved in the essential processes of crossing over and mismatch repair of DNA and in cells that undergo a dramatic alteration in gene expression. However, contributions of this linker histone to these processes are unknown. Subtle differences are found in the H1t promoter compared to the other H1 promoters. Nevertheless, several lines of evidence support the hypothesis that a sequence element designated TE that is located within the H1t promoter is essential for enhanced testis-specific transcription of this gene. Transgenic mice bearing a rat H1t transgene which contains a replacement of the TE element with stuffer DNA fail to express rat H1t mRNA. In addition, an upstream sequence appears to function as a silencer element that leads to transcriptional repression of the H1t gene in nongerminal cells. Thus, multiple promoter elements appear to contribute to regulation of transcription of the histone H1t gene.

Binding of nuclear proteins to a conserved histone H1t promoter element suggests an important role in testis-specific transcription.

The testis-specific histone H1t gene is transcribed only in primary spermatocytes during spermatogenesis. Recently, expression of the rat gene was shown to be limited to primary spermatocytes in transgenic mice, revealing that promoter elements sufficient for regulating tissue-specific transcription were present in the cloned rat gene. In this study the mouse histone H1t gene has been cloned, and sequenced and its promoter region has been compared to the rat H1t promoter with regard to conserved elements and protein binding activity. The amino acid sequence of each of the three H1t coding region domains is conserved when compared to the homologous domain in H1t derived from other species. H1t mRNA is found only in testis, where it accumulates to a high steady-state level, and examination of enriched testis cell populations shows that expression is limited to primary spermatocytes. Protein binding assays using nuclear extracts from various mouse tissues reveal testis-specific binding to TE1 and TE2, imperfect inverted repeat elements within the larger TE element. Although the H1t promoter contains an Sp1 consensus motif within the H1t/TE element, binding of testis Sp1 to the motif could not be detected using specific anti-Sp1 antibodies.

Expression of the testis-specific histone H1t gene: evidence for involvement of multiple cis-acting promoter elements.

The histone H1t gene is expressed exclusively in testis primary spermatocytes. Previous studies indicate that accumulation of H1t mRNA occurs only in primary spermatocytes in normal rats and in transgenic mice bearing the rat H1t transgene. In this study, DNA sequences of human, monkey, mouse, and rat H1t genes were compared and found to be almost identical in the proximal promoter region extending from the H1/AC box through the TATAA box. In addition to conserved elements common to replication-dependent H1 promoters, the H1t promoter contains a unique TE element, and sequences within this element may contribute to enhanced expression of the gene in primary spermatocytes. Two imperfect inverted repeat sequences designated TE1 and TE2, that are located within the larger TE element, overlap a central GC-rich region and bind specifically to nuclear proteins derived from primary spermatocytes. Protein interactions characterized by methylation interference and UV cross-linking experiments indicate that a complex of proteins with a molecular mass of approximately 180 kDa binds TE1. The GC-rich region in H1t and in some replication dependent histone H1 promoters contains an Sp1 consensus sequence. Although the H1t/TE element that contains the GC-rich region binds nuclear proteins, it does not appear to bind Sp1 obtained from cell populations enriched in primary spermatocytes as determined by electrophoretic mobility supershift assays using polyclonal anti-Sp1 antibodies.

Testis-specific expression of the rat histone Hlt gene in transgenic mice.

The testis-specific histone Hlt gene is transcribed only in testis. The appearance of testis-specific nuclear proteins that bind to a unique promoter sequence element designated Hlt/TE located between the H1/AC box and the H1/CCAAT box correlates with the onset of transcription of the Hlt gene during the meiotic cell cycle. In order to determine whether sequences flanking the rat Hlt gene are sufficient to confer tissue-specific expression in vivo, a 6859 bp EcoRI restriction fragment of genomic DNA containing the rat histone Hlt gene has been microinjected into mouse embryos. S1 nuclease protection analysis has shown that the descendants of the resulting transgenic mice express the rat gene in the proper tissue and at the proper meiotic cell cycle stage. Furthermore, when populations of mouse testis cells were prepared by centrifugal elutriation, only the fraction enriched in pachytene primary spermatocytes had a significant steady-state level of rat Hlt mRNA. Although the copy-number of the transgene was variable in these animals, rat Hlt mRNA levels in high copy-number animals never exceeded 2.6 times the level in normal rat testes. The appearance of appropriate meiotic cell cycle-specific transcription indicates the importance of the conserved promoter sequence elements between the two species.

Characterization of proteoglycans produced by rat pleural mesothelial cells in vitro.

The mesothelial cell envelopes the surface of the parietal and visceral pleura. These cells are known to synthesize most of the protein constituents of the pleural basement membrane and interstitium. This study examined the ability of a rat pleural mesothelial cell line to synthesize proteoglycans in vitro. Cells were labeled with inorganic 35SO4 to label the glycosaminoglycan moiety of proteoglycans. The medium and combined cell membrane/extracellular matrix fractions contained 73 and 25% of the proteoglycan radioactivity, respectively. The medium contained a single chondroitin/dermatan sulfate proteoglycan of approximately 190 kDa, consistent with biglycan. As determined by Northern analysis of steady-state levels of messenger RNA, the cells contained message for biglycan. Stimulation of the cells with epidermal growth factor resulted in the appearance of a second chondroitin/dermatan sulfate proteoglycan of approximately 97 kDa, characteristic of decorin. The cell membrane/matrix contained a biglycan-like chondroitin/dermatan proteoglycan and several heparan sulfate proteoglycans. Pleural mesothelial cells in vitro are capable of synthesizing a variety of interstitial and basement membrane proteoglycans.

Primate testicular histone H1t genes are highly conserved and the human H1t gene is located on chromosome 6.

The testis-specific histone H1t gene is known to be transcribed only in pachytene primary spermatocytes during spermatogenesis. Previous studies of the rat histone H1t gene revealed a unique promoter sequence element between the H1/GC box and the H1/CCAAT box. Proteins in crude nuclear extracts of rat testis bind specifically to this sequence element and a temporal correlation exists between the appearance of these DNA binding proteins and the onset of transcription. These discoveries led to a search for histone H1t genes in other mammalian species. The human and monkey histone H1t genes were amplified from genomic DNA using the polymerase chain reaction (PCR). The amplified genes were cloned and the genomic derived inserts were sequenced using linear PCR. Both proximal promoters contained the highly conserved H1/AC box, H1/CCAAT box, and H1/TATA box found in nongerminal H1 genes. Both promoters also contained the H1/GC box and the H1t/CCTAGG sequence element between the H1/GC box and H1/CCAAT box previously seen only in the H1t promoter. Specific amplification of the human H1t gene using template DNA samples from a NIGMS human/rodent somatic cell hybrid mapping panel has shown that the human histone H1t gene is located on chromosome 6.

Histone H1t: a tissue-specific model used to study transcriptional control and nuclear function during cellular differentiation.

One of the most prominent and best studied family of genes is the histone gene family. In recent years, histone gene regulation during the cell cycle of somatic cells has been studied extensively. This paper is intended to highlight and emphasize recent data concerning the tissue-specific expression of histone H1t using spermatogenesis as a model system. In this article we describe a unique DNA element within the proximal promoter of the histone H1t gene. This element has been shown to bind exclusively to nuclear proteins from pachytene spermatocytes and early spermatids. Thus, there is a strong temporal correlation between the appearance of the testis-specific DNA-binding protein and the onset of transcription of the testis-specific histone H1t gene.

Pleural mesothelial cell response to inflammation: tumor necrosis factor-induced mitogenesis and collagen synthesis.

This study examined the effects of tumor necrosis factor-alpha on pleural mesothelial cell proliferation and collagen synthesis, functions which may be important in the response of the pleura to injury. Tumor necrosis factor-alpha caused a significant increase in proliferation and collagen production by rat pleural mesothelial cells in vitro. Proliferation increased in a time- and dose-dependent manner, resulting in an approximate twofold increase in the uptake of [3H]thymidine relative to control. The uptake of [3H]proline into collagenase-sensitive protein increased in a dose-dependent manner for concentrations of tumor necrosis factor-alpha > or = 1.0 ng/ml. The increase in collagen production were associated with increased steady-state levels of alpha 1(I)-procollagen mRNA. These results suggest that tumor necrosis factor-alpha may have a significant effect on pleural mesothelial cell function in vivo in the setting of inflammation. Increases in pleural mesothelial cell proliferation and collagen synthesis in response to inflammatory mediators, like tumor necrosis factor-alpha, may be important in healing the pleura after injury by a variety of disease processes.

Indolent orbital apex syndrome caused by occult mucormycosis.

The chronic or indolent presentation of rhino-orbital mucormycosis, as defined by the presence of symptoms for more than 1 month before diagnosis, is extremely unusual. A 45-year-old man with stable diabetes presented with a right orbital apex syndrome and minimal ethmoid and sphenoid sinusitis. Progression was indolent, and the diagnosis was not made until 7 weeks after admission, when a third biopsy was prompted by new cavernous sinus and carotid artery thromboses. Mucormycosis was found. The patient improved on amphotericin B (2 g) and strict blood glucose control. A remarkable aberrant regeneration of the right oculomotor nerve was seen following treatment. He remains free of active disease 4 years later. Orbital symptoms in well-controlled diabetics, which may even remain stable for weeks and lack direct signs of tissue invasion, should raise the suspicion of mucormycosis.

Tissue-specific binding of testis nuclear proteins to a sequence element within the promoter of the testis-specific histone H1t gene.

The rat histone H1t gene is transcribed only in testis germinal cells. This testis-specific chromosomal protein is first synthesized during spermatogenesis in pachytene spermatocytes and the entire complement of testis histones is replaced during the midspermatid stage of spermiogenesis by positively charged transition nuclear proteins TP1 and TP2. Mobility shift assays conducted using crude nuclear protein extracts from different tissues and an 18-bp DNA sequence element within the H1t promoter as a probe reveal binding only with nuclear proteins from testis. The binding is specifically competed with an excess of the same unlabeled DNA fragment but not with heterologous competitors. A larger oligonucleotide corresponding to the same sequence element plus 18 bp of the adjacent downstream H1/CCAAT element binds nuclear proteins from all tissues tested, but a unique low mobility band is formed only with testis extracts. Protein-DNA crosslinking experiments reveal that two major polypeptides with molecular weights of approximately 13 and 30 kDa bind to the 18-bp H1t promoter sequence element. This strong correlation between the tissue where the H1t gene is transcribed and the presence of testis-specific nuclear proteins that bind to a sequence element within the testis histone H1t promoter supports the possibility that these DNA-binding proteins may participate in formation of an active transcription initiation complex with the testis H1t promoter.

Temporal correlation between the appearance of testis-specific DNA-binding proteins and the onset of transcription of the testis-specific histone H1t gene.

The histone H1t gene is transcribed only in testis. Northern blot analyses reveal that transcription of the H1t gene occurs first in pachytene primary spermatocytes. Thus, there is a temporal correlation between onset of transcription of the gene and synthesis of histone H1t in primary spermatocytes during spermatogenesis. Previous studies revealed that replacement of most H1t and core histones occurs during the midspermatid stage of spermiogenesis by transition proteins TP1 and TP2. In this paper we extend our study of the specific binding of testis nuclear proteins to a unique sequence element within the H1t promoter. The relatively tight binding is competed with an excess of homologous DNA but not with a mutated element. Testis proteins from prepubertal animals do not bind to the 18-bp promoter element out proteins from enriched populations of primary spermatocytes do bind. Therefore, the temporal correlation between onset of transcription of the H1t gene and the time when the specific H1t promoter-binding proteins are detected in primary spermatocytes suggests that the DNA-binding proteins might be germinal cell-specific transcription factors that participate in formation of an active H1t transcription initiation complex. These studies present the first analysis of binding sites for testis nuclear proteins from primary spermatocytes within the promoter of a gene expressed only during this stage of spermatogenesis.