Cytosine methylation represses glutathione S-transferase P1 (GSTP1) gene expression in human prostate cancer cells.

Methylation of the glutathione S-transferase P1 (GSTP1) gene has been described as a highly specific and sensitive biomarker for prostate cancer. However, at present, it is not known whether methylation represses GSTP1 gene expression in human prostate cancer. We found the GSTP1 gene promoter to be completely methylated in the LNCaP prostate cancer cell line, where this gene is transcriptionally inactive. In contrast, Du145 and PC3 prostate cancer cells express the GSTP1 gene and exhibit methylated and unmethylated GSTP1 alleles. In a transient transfection assay using LNCaP cells, methylation of the GSTP1 promoter-driven luciferase reporter vector (GSTP1-pGL3) resulted in a >20-fold inhibition of transcription, and this repression was not relieved by the presence of a histone deacetylase inhibitor, trichostatin A (TSA). Treatment of LNCaP cells with a DNA methyltransferase inhibitor, 5-Aza-2'-deoxycytidine, resulted in demethylation and activation of the GSTP1 gene. In contrast, TSA treatment failed to demethylate or activate the GSTP1 gene. Fully methylated but not unmethylated GSTP1 promoter fragment was shown to bind to a complex similar to methyl cytosine-binding protein complex 1 that contains methyl-CpG-binding domain 2 protein (MBD2) in electrophoretic mobility shift assays using LNCaP cell nuclear extracts. These data demonstrate that cytosine methylation can repress GSTP1 gene expression in LNCaP prostate cancer cells and that this effect is possibly mediated by a methyl cytosine-binding protein complex 1-like complex. Furthermore, these data also support the notion of the dominance of methylation over TSA-sensitive histone deacetylation in silencing genes with a high CpG density in the promoter region.

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.

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.

Synthesis and secretion of proteins by postpartum human oviductal tissue in culture.

An explant culture system that used labelled leucine and two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) with fluorography was used to identify specific de novo synthesized and released polypeptides by the human postpartum oviduct. Both ampulla and isthmus tissue in culture exhibited de novo synthesis and release of a large number of polypeptide subunits. Immunoglobulins A and G appear to be the major proteins produced in the ampulla. In addition, two complexes of acidic (pI less than 5) polypeptide subunits are found primarily in ampulla culture medium. Two families of proteins (Mr 51,000 and 60,000) are released by the isthmus but appear to be minor in the ampulla cultures.