Sidney R. Grimes

Upregulation of prostate specific membrane antigen/folate hydrolase transcription by an enhancer.

Prostate specific membrane antigen (PSMA), also known as folate hydrolase (FOLH1), is a 100 kDa glycoprotein with elevated expression in prostate epithelial tissue. Expression of PSMA is upregulated as prostate tumor grade increases and is found in the vasculature of many tumors, with no presence in benign tissues. Due to the potential of the regulatory elements of the PSMA promoter and enhancer to be used in gene therapy and as biomarkers for prostate cancer under conditions of androgen ablation during treatment, we sequenced and analyzed the ability of 5.5 kb of PSMA promoter/leader region to promote transcription. A recently discovered enhancer, found in the third intron of the PSMA gene, FOLH1, was also studied. The promoter/leader region sequence provided basal expression in transcription assays, while addition of the enhancer activated transcription 41-fold in transient transfections and 144-fold in stable transfections of the LNCaP prostate cell line. This enhancement of transcription was not found in nonprostate cell lines or prostate cell lines that do not express PSMA. An analysis of the ability of androgens to act via the PSMA promoter/leader region and enhancer to activate transcription in transiently transfected LNCaP cells revealed no significant androgen response using the FOLH1 promoter/leader region and a downregulation of 42% with addition of the enhancer. In stably transfected LNCaP cells, the FOLH1 promoter/leader region produced a 21% downregulation in response to androgens, while addition of the enhancer resulted in a 45% downregulation. These results demonstrate significant upregulation of transcription by the PSMA promoter/enhancer, with specificity for the LNCaP prostate cell line, and downregulation of transcription in response to physiological levels of androgen.

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.

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.

Regulatory factor X2 (RFX2) binds to the H1t/TE1 promoter element and activates transcription of the testis‐specific histone H1t gene

Transcription of the mammalian testis‐specific linker histone H1t gene occurs only in pachytene primary spermatocytes during spermatogenesis. Studies of the wild type (Wt) and mutant H1t promoters in transgenic mice show that transcription of the H1t gene is dependent upon the TE promoter element. We purified an 85 kDa protein from rat testis nuclear extracts using the TE1 subelement as an affinity chromatography probe and analysis revealed that the protein was RFX2. The TE1 element is essentially an X‐box DNA consensus element and regulatory factor X (RFX) binds specifically to this element. Polyclonal antibodies directed against RFX2 supershift the low mobility testis nuclear protein complex formed in electrophoretic mobility shift assays (EMSA). RFX2 derived from primary spermatocytes, where the transcription factor is relatively abundant, binds with high affinity to the TE1 element. Coexpression of RFX2 together with an H1t promoter/reporter vector activates the H1t promoter in a cultured GC‐2spd germinal cell line, but mutation of either the TE1 subelement or the TE2 subelements represses activity. These observations lead us to conclude that the TE1 and TE2 subelements of the testis‐specific histone H1t promoter are targets of the transcription factor RFX2 and that this factor plays a key role in activating transcription of the H1t gene in primary spermatocytes. Published 2003 Wiley‐Liss, Inc.

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.

Testis-Specific Histone H1t Gene Is Hypermethylated in Nongerminal Cells in the Mouse

Abstract 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. J. Cell. Biochem. 75:555–565, 1999. Published 1999 Wiley‐Liss, Inc.

Transcription factor RFX2 is abundant in rat testis and enriched in nuclei of primary spermatocytes where it appears to be required for transcription of the testis-specific histone H1t gene.

Previous work in our laboratory revealed upregulated transcription of the testis‐specific linker histone H1t gene in pachytene primary spermatocytes during spermatogenesis. Using the H1t X‐box as an affinity chromatography probe, we identified Regulatory Factor X2 (RFX2), a member of the RFX family of transcription factors, as a nuclear protein that binds the probe. We also showed that RFX2 activated the H1t promoter in transient expression assays. However, other RFX family members have the same DNA‐binding domain and they also may regulate H1t gene expression. Therefore, in this study we examined the distribution of RFX2 and other RFX family members in rat testis germinal cells and in several tissues. Among tissues examined, RFX2 is most abundant in testis. Testis RFX2 is most abundant in spermatocytes where transcription of the H1t gene is upregulated and the steady‐state H1t mRNA level is high. RFX2 levels decrease but RFX1 levels increase in early spermatids where H1t gene transcription is downregulated. Antibodies against RFX2 generate a shifted band in electrophoretic mobility shift assays (EMSA) using H1t or testisin X‐box DNA probes with nuclear proteins from spermatocytes. These data support the hypothesis that RFX2 expression is upregulated in spermatocytes where it participates in activating transcription of the H1t gene and other testis genes. These data also support the possibility that other RFX family members may bind to the H1t promoter in other testis germinal cell types and in nongerminal cells to downregulate H1t gene transcription. J. Cell. Biochem. 99: 735–746, 2006.

Transcriptional repression of the testis-specific histone H1t gene mediated by an element upstream of the H1/AC box.

The testis-specific histone H1t gene is transcribed exclusively in primary spermatocytes and may be important for chromatin structure, transcription, and DNA repair during this stage of spermatogenesis. Transcriptional repression of the gene in other cell types is mediated in part by specific proximal and distal promoter elements and in some cell types by methylation of CpG dinucleotides within the promoter. Our laboratory identified a distal promoter element located between 948 and 780 bp upstream from the transcription initiation site and another laboratory identified a GC-rich region between the TATA box and transcription initiation site that contribute to repression. In this article we address transcriptional repression of the histone H1t gene by an element within the proximal promoter. We report discovery of an element designated H1t promoter repressor element (RE) located between -130 and -106 bp that contributes to repression. The findings support the hypothesis that multiple mechanisms are involved in transcriptional repression of the H1t gene. Transcriptional repression mediated by the RE element in NIH 3T3 cells appears to differ significantly from the mechanism mediated by the GC-rich region. Furthermore, binding proteins that form the RE complex are not present in rat testis where the gene is actively transcribed. Our findings provide a molecular basis for histone H1t gene repression.

Transcription factor RFX4 binding to the testis‐specific histone H1t promoter in spermatocytes may be important for regulation of H1t gene transcription during spermatogenesis

The X‐box binding protein RFX4 is highly expressed in testis in contrast with other tissues, but its function there is unknown. Another family member abundant in testis, RFX2, has been shown to bind to the X‐Box elements in the promoter of the testis specific histone H1t, which is expressed only in pachytene spermatocytes. RFX proteins are known to dimerize, and there is the possibility that the abundant testis RFX4, which is also expressed in pachytene spermatocytes as shown by RT‐PCR and Western blotting, may interact with RFX2 in these cells. In EMSA anti‐RFX2 polyclonal antibodies produce a supershifted complex with testis extracts and an X‐Box probe. On the other hand, RFX4 polyclonal antibodies do not supershift the complex but appear to enhance formation of the complex. RFX4 appears to co‐precipitate with RFX2 in immunoprecipitation, and to co‐purify with RFX2 in an affinity purification using a biotinylated X‐box affinity probe. In ChIP assays RFX4 also binds to the H1t promoter in vivo. These data suggest a possible regulatory role for RFX4 in transcription of the histone H1t gene during spermatogenesis. J. Cell. Biochem. 105: 61–69, 2008.