Tumor-suppression function of transcription factor USF2 in prostate carcinogenesis.

Although the transcription factor USF2 has been implicated in the regulation of cellular growth and proliferation, it is unknown whether alterations in USF2 contribute to tumorigenesis and tumor development. We examined the role of USF2 in prostate tumorigenesis. Western blot analysis revealed markedly decreased USF2 levels in three androgen-independent prostate cancer cell lines, PC-3, DU145, and M12, as compared to nontumorigenic prostate epithelial cells or the androgen-dependent cell line, LNCaP. Ectopic expression of USF2 in PC-3 cells did not affect the cell proliferation rate of PC-3 cells on plastic surfaces. However, it dramatically decreased anchorage-independent growth of PC-3 cells in soft agar (90-98% inhibition) and the invasion capability (80% inhibition) of PC-3 cells in matrix gel assay. Importantly, expression of USF2 in PC-3 cells inhibited the tumorigenicity of PC-3 cells in an in vivo nude mice xenograft model (80-90% inhibition). These results suggest that USF2 has tumor-suppression function. Consistent with its function in tumor suppression, we found that the USF2 protein is present in normal prostate epithelial cells but absent in 18 of 42 (43%) human prostate cancer tissues (P = 0.015). To further examine the functional role of USF2 in vivo, we generated mice with genetic deletion of USF2 gene. We found that USF2-null mice displayed marked prostate hyperplasia at a young age, suggesting that USF2 is involved in the normal growth and differentiation of prostate. Together, these studies demonstrate that USF2 has tumor-suppressor function and plays a role in prostate carcinogenesis.

Spatial organization of RNA polymerase II transcription in the nucleus.

In eukaryotic cells, mRNA synthesis is carried out by large, multifunctional complexes that are also involved in coordinating transcription with other nuclear processes. This survey focuses on the distribution and structural arrangement of these complexes within the nucleus, in relationship with the discrete positioning of particular chromosomal loci. To better understand the link between the spatial organization of the nucleus and the regulation of gene expression, it is necessary to combine information from biochemical studies with results from microscopic observations of preserved nuclear structures. Recent experimental approaches have made this possible. The subnuclear locations of specific chromosome loci, RNA transcripts, RNA polymerases, and transcription and pre-mRNA-processing factors can now be observed with computer-assisted microscopy and specific molecular probes. The results indicate that RNA polymerase II (RNAPII) transcription takes place at discrete sites scattered throughout the nucleoplasm, and that these sites are also the locations of pre-mRNA processing. Transcribing polymerases appear to be grouped into clusters at each transcription site. Cell cycle-dependent zones of transcription and processing factors have been identified, and certain subnuclear domains appear specialized for expression or silencing of particular genes. The arrangement of transcription in the nucleus is dynamic and depends on its transcriptional activity, with the RNAPII itself playing a central role in marshalling the large complexes involved in gene expression.

Multiple rounds of transcription by RNA polymerase II at covalently cross-linked templates.

An important control point for gene regulation is the frequency of initiations leading to different numbers of RNA polymerases simultaneously transcribing the same gene. To date, the only direct assay for multiple-round transcription by RNA polymerase II in vitro required G-free cassette-containing templates and GTP-free conditions and was thus restricted in application. Here we used instead templates containing a triplex-directed interstrand psoralen-DNA cross-link to block RNA polymerase II elongation at a specific location. Covalently cross-linked templates allowed simultaneous detection of both specific initiation and reinitiation with any combination of promoter and transcribed sequence. In reconstituted systems, identical stacking of RNA polymerases was observed when the first polymerase was halted by GTP deprivation at the end of a G-free cassette or by a covalent cross-link downstream of different transcribed sequences. In contrast to transcription of G-free cassettes, reinitiation was unaffected by the transcription factor SII on sequences containing all four nucleotides. In crude nuclear extracts, transcription of covalently cross-linked templates yielded a reinitiation pattern with a wider spacing than in more purified fractions, indicating that the elongation complexes from nuclear extract contained a different form of RNA polymerase II or a different complement of associated factors.

Ion channel properties of a protein complex with characteristics of a glutamate/N-methyl-D-aspartate receptor.

The functional reconstitution of glutamate receptor proteins purified from mammalian brain has been difficult to accomplish. However, channels activated by L-glutamate (L-Glu) and N-methyl-D-aspartate (NMDA) were detected in planar lipid bilayer membranes (PLMs) following the reconstitution of a complex of proteins with binding sites for NMDA receptor (NMDAR) ligands. The presence of glycine was necessary for optimal activation. A linear current-voltage relationship was observed with the reversal potential being zero. Channels activated by L-Glu had conductances of 23, 47 and 65 pS, and were suppressed partially by competitive and fully by noncompetitive inhibitors of NMDARs. Magnesium had little effect on the reconstituted channels.

Sarkosyl block of transcription reinitiation by RNA polymerase II as visualized by the colliding polymerases reinitiation assay.

There are indications that different concentrations of Sarkosyl can block transcription initiation by RNA polymerase II in vitro at different functional steps [Hawley and Roeder (1985) J. Biol. Chem. 260, 8163-8172]. Consequently, this reagent could be a very useful tool for mechanistic studies. So far, however, evidence for the selectivity of Sarkosyl effects on RNA polymerase II transcription has been only indirect. To directly investigate the effect of Sarkosyl on transcription initiation and reinitiation by RNA polymerase II, we employed the reinitiation assay based on utilization of templates containing G-free cassettes (colliding polymerases reinitiation assay, or CoPRA). These experiments showed unambiguously that, under the appropriate conditions, Sarkosyl can be used to block transcription reinitiation by RNA polymerase II while allowing a first round of initiations from preassembled initiation complexes. This inhibition is not due to a disruption of the SII-dependent elongation of the reinitiated transcripts, and the levels of Sarkosyl that prevent transcription reinitiation coincide with the levels that block preinitiation complex assembly. However, Sarkosyl addition to transcription reactions reconstituted with partially purified transcription factors was found to have several undesirable side effects. The usefulness and limitations of the Sarkosyl-based and CoPRA assays for measurements of transcription reinitiation are discussed.

Synthesis of reinitiated transcripts by mammalian RNA polymerase II is controlled by elongation factor SII.

Previous studies have revealed that the in vitro synthesis of reinitiated transcripts by RNA polymerase II requires an additional activity, designated reinitiation transcription factor (RTF), which is distinct from all of the general class II initiation factors. While further characterizing this activity, it was found that RTF displays properties indistinguishable from those of the RNA polymerase II elongation factor SII. In addition, Western blot analysis using SII-specific antibodies revealed that human SII is a major component in purified RTF preparations. The functional equivalence of the two proteins was established using recombinant SII, which proved fully capable of substituting for RTF in the reinitiation assay. In these reconstituted reactions, transcription complexes resulting from reinitiation events required SII to proceed through a 400 bp G-free cassette, while complexes resulting from the first round of initiations were SII-independent. Reinitiations can take place in the absence of SII; however, addition of the elongation factor is essential for full extension of the reinitiated transcripts. These results suggest that events taking place at the promoter (e.g. first-round initiations versus reinitiations) can create marked differences in the properties of RNA polymerase II elongation complexes.

Transcription factor requirement for multiple rounds of initiation by human RNA polymerase II.

We have investigated conditions that allow multiple rounds of transcription initiation from the adenovirus major late promoter in an in vitro system derived from HeLa cell nuclear extracts. Templates containing guanine-free cassettes provided a direct assay for discriminating between reinitiated transcripts and transcripts generated by a first-round of transcription initiations. When reactions were reconstituted with the previously characterized class II transcription factors (TFIIA, TFIIB, TFIID, TFIIE/F), transcription by human RNA polymerase II from the adenovirus major late promoter was essentially restricted to a single round of initiations. Reinitiations at previously transcribed major late templates required an additional activity, designated reinitiation transcription factor (RTF). The RTF activity could be separated from the required transcription initiation factors. Semipurified human RTF also promoted transcription reinitiations at minimal promoters derived from the human c-myc, histone H4, and heat shock 70-kDa protein genes, indicating that the same reinitiation factor may be utilized by many, if not all, genes. The possible role of RTF in regulating the transcription rate of various class II genes is discussed.

Luminescence probe studies of ionomers-II Steady-state measurements from sulphonated polyethylene and teflon membranes.

Luminescence probe studies of sulphonated Teflon and sulphonated polyethylene ionomer membranes are described. These studies have shown that the micropolarities of the ionic clusters within these ionomers are quite dynamic, varying with the nature of the chain material and the membrane water content. These studies also suggest that polymer chain material intrudes into the ionic cluster phase.