Drosophila C-terminal binding protein functions as a context-dependent transcriptional co-factor and interferes with both mad and groucho transcriptional repression.

Drosophila C-terminal binding protein (dCtBP) and Groucho have been identified as Hairy-interacting proteins required for embryonic segmentation and Hairy-mediated transcriptional repression. While both dCtBP and Groucho are required for proper Hairy function, their properties are very different. As would be expected for a co-repressor, reduced Groucho activity enhances the hairy mutant phenotype. In contrast, reduced dCtBP activity suppresses it. We show here that dCtBP can function as either a co-activator or co-repressor of transcription in a context-dependent manner. The regions of dCtBP required for activation and repression are separable. We find that mSin3A-histone deacetylase complexes are altered in the presence of dCtBP and that dCtBP interferes with both Groucho and Mad transcriptional repression. Similar to CtBP's role in attenuating E1A's oncogenicity, we propose that dCtBP can interfere with corepressor-histone deacetylase complexes, thereby attenuating transcriptional repression. Hairy defines a new class of proteins that requires both CtBP and Groucho co-factors for proper function.

Promoter-specific targeting of human SWI-SNF complex by Epstein-Barr virus nuclear protein 2.

The multiprotein human SWI-SNF (hSWI-SNF) complex is a chromatin-remodeling machine that facilitates transcription by overcoming chromatin-mediated gene repression. We had previously shown that hSNF5/INI1, an intrinsic, consistent component of the hSWI/SNF complex, is associated with Epstein-Barr nuclear antigen 2 (EBNA2) and have proposed that EBNA2 directs this complex to key EBNA2-responsive viral and cellular genes. Using chromatin immunoprecipitation and quantitative PCR, we show that antibodies directed against components of the hSWI-SNF complex preferentially precipitate chromatin-associated DNA that contains a targeted EBNA2-responsive element in the context of both episomal and cellular chromatin. This enrichment does not occur in EBNA2-negative cells or when the EBNA2-responsive element is mutated. The stable association of the hSWI-SNF complex with the EBNA2-responsive promoter can also be disrupted by deletion of the TATA element, suggesting that EBNA2 in itself is insufficient to mediate stable targeting of the hSWI-SNF complex. These results demonstrate that recruitment of the hSWI-SNF complex to selected promoters can occur in vivo through its interaction with site-specific activator proteins and that stable targeting may require the presence of basal transcription factors.

Long-distance transcriptional enhancement by the histone acetyltransferase PCAF.

Enhancers are defined by their ability to stimulate gene activity from remote sites and their requirement for promoter-proximal upstream activators to activate transcription. Here we demonstrate that recruitment of the p300/CBP-associated factor PCAF to a reporter gene is sufficient to stimulate promoter activity. The PCAF-mediated stimulation of transcription from either a distant or promoter-proximal position depends on the presence of an upstream activator (Sp1). These data suggest that acetyltransferase activity may be a primary component of enhancer function, and that recruitment of polymerase and enhancement of transcription are separable. Transcriptional activation by PCAF requires both its acetyltransferase activity and an additional activity within its N terminus. We also show that the simian virus 40 enhancer and PCAF itself are sufficient to counteract Mad-mediated repression. These results are compatible with recent models in which gene activity is regulated by the competition between deacetylase-mediated repression and enhancer-mediated recruitment of acetyltransferases.

The immunoglobulin heavy chain locus control region increases histone acetylation along linked c-myc genes.

In chromosome translocations characteristic of Burkitt lymphomas (BL) and murine plasmacytomas, c-myc genes become juxtaposed to immunoglobulin heavy-chain (IgH) sequences, resulting in aberrant c-myc transcription. Translocated c-myc alleles that retain the first exon exhibit increased transcription from the normally minor c-myc promoter, P1, and increased transcriptional elongation through inherent pause sites proximal to the major c-myc promoter, P2. We recently demonstrated that a cassette derived from four DNase I-hypersensitive sites (HS1234) in the 3'Calpha region of the IgH locus functions as an enhancer-locus control region (LCR) and directs a similar pattern of deregulated expression of linked c-myc genes in BL and plasmacytoma cell lines. Here, we report that the HS1234 enhancer-LCR mediates a widespread increase in histone acetylation along linked c-myc genes in Raji BL cells. Significantly, the increase in acetylation was not restricted to nucleosomes within the promoter region but also was apparent upstream and downstream of the transcription start sites as well as along vector sequences. Histone hyperacetylation of control c-myc genes, which was induced by the deacetylase inhibitor trichostatin A, mimics the effect of the HS1234 enhancer on expression from the c-myc P2 promoter, but not that from the P1 promoter. These results suggest that the HS1234 enhancer stimulates transcription of c-myc by a combination of mechanisms. Whereas HS1234 activates expression from the P2 promoter through a mechanism that includes increased histone acetylation, a general increase in histone acetylation is not sufficient to explain the HS1234-mediated activation of transcription from P1.

Post-transcriptional regulation of vascular endothelial growth factor mRNA by the product of the VHL tumor suppressor gene.

The VHL tumor suppressor gene is inactivated in patients with von Hippel-Lindau disease and in most sporadic clear cell renal carcinomas. Although VHL protein function remains unclear, VHL does interact with the elongin BC subunits in vivo and regulates RNA polymerase II elongation activity in vitro by inhibiting formation of the elongin ABC complex. Expression of wild-type VHL in renal carcinoma cells with inactivated endogenous VHL resulted in unaltered in vitro cell growth and decreased vascular endothelial growth factor (VEGF) mRNA expression and responsiveness to serum deprivation. VEGF is highly expressed in many tumors, including VHL-associated and sporadic renal carcinomas, and it stimulates neoangiogenesis in growing solid tumors. Despite 5-fold differences in VEGF mRNA levels, VHL overexpression did not affect VEGF transcription initiation or elongation as would have been suggested by VHL-elongin association. These results suggest that VHL regulates VEGF expression at a post-transcriptional level and that VHL inactivation in target cells causes a loss of VEGF suppression, leading to formation of a vascular stroma.

HMG17 is a chromatin-specific transcriptional coactivator that increases the efficiency of transcription initiation.

We have examined the effect of HMG17 on transcription by RNA polymerase II by the assembly and analysis of HMG17-containing chromatin templates consisting of regularly spaced nucleosomal arrays. Structural analysis of the chromatin indicated that HMG17 is incorporated into chromatin in a physiological manner with the full complement of core histones. The transcriptional studies revealed that HMG17 stimulates transcription in conjunction with the sequence-specific activator GAL4-VP16. This effect was observed with chromatin, but not with non-nucleosomal templates, and required the presence of HMG17 during chromatin assembly. The incorporation of HMG17 into chromatin resulted in a 7- to 40-fold stimulation of GAL4-VP16-activated transcription to levels that were comparable to those observed with histone-free DNA templates. In contrast, transcription from HMG17-containing chromatin was not detectable in the absence of GAL4-VP16 or with a GAL4 derivative [GAL4(1-147)] lacking the VP16 activation domain. Finally, the incorporation of HMG17 into chromatin was found to increase the efficiency of transcription initiation, but not the extent of transcriptional elongation. Thus, HMG17 is a chromatin-specific transcriptional coactivator that increases the efficiency of initiation of transcription by RNA polymerase II.

Sequences within the coding regions of clotting factor VIII and CFTR block transcriptional elongation.

The clotting factor VIII (FVIII) and cystic fibrosis transmembrane conductance regulator (CFTR) cDNAs have dramatically reduced levels of expression compared to clotting factor IX (FIX) and other cDNAs (100 and 1,000-fold lower, respectively), when produced in cells by using an expression vector. Part of the inhibitory signal in the FVIII cDNA has been localized to a 1.2-kb inhibitory sequence (FVIII INS), which decreased steady-state RNA levels from a retroviral vector by 30- to 100-fold. An analysis of RNA degradation indicated that the FVIII INS vector RNA is relatively stable. Nuclear run-on experiments with the FVIII INS vector demonstrated a low signal for FVIII, in contrast to the high signal for a FIX vector. The low signal for FVIII INS was not due to a decrease in transcriptional initiation. Thus, FVIII expression is reduced through a block to transcriptional elongation, as has been found in c-myc and other genes. We show that the inhibitory effect of FVIII INS is orientation dependent with regard to the promoter. In addition, the inhibitory effect is position dependent, because expression of FVIII INS sequence increased when it was moved 1 kb further from the promoter in a retroviral vector. Similar results were observed by using a retroviral vector for expression of the CFTR cDNA. The CFTR retroviral vector produced 1,000-fold decreased steady-state RNA levels, compared to the parent vector. Nuclear run-on analysis with the CFTR vector revealed a block to transcriptional elongation within the CFTR cDNA. The presence of blocks to transcriptional elongation within the FVIII and CFTR cDNAs complicates efforts to produce high levels of these proteins for therapeutic purposes and to develop high-titer retroviral expression vectors for human gene therapy.

Promoter-proximal pausing of RNA polymerase II defines a general rate-limiting step after transcription initiation.

We have shown previously that the majority of RNA polymerase II complexes initiated at the c-myc gene are paused in the promoter-proximal region, similar to observations in the Drosophila hsp70 gene. Our analyses define the TATA box or initiator sequences in the c-myc gene as necessary components for the establishment of paused RNA polymerase II. Deletion of upstream sequences or even the TATA box does not influence significantly the degree of transcriptional initiation or pausing. Deletion of both the TATA box and sequences at the transcription initiation site, however, abolishes transcriptional pausing of transcription complexes but still allows synthesis of full-length RNA. Further analyses with synthetic promoter constructs reveal that the simple combination of upstream activator with TATA consensus sequences or initiator sequences act synergistically to recruit high levels of RNA polymerase II complexes. Only a minor fraction of these complexes escapes into regions further downstream. Several different trans-activation domains fused to GAL4-DNA-binding domains, including strong activators such as VP16, do not eliminate promoter-proximal pausing of RNA polymerase. Thus, we conclude that pausing of RNA polymerase II is a common phenomenon in eukaryotic transcription and does not require complex promoter structures. Further analyses reveal that enhancers have a modest influence on transcription initiation and on release of transcription complexes out of the pause site but may function primarily to increase the elongation competence of transcription complexes.

Common mechanisms for the control of eukaryotic transcriptional elongation.

Regulation of transcriptional elongation is emerging as an important control mechanism for eukaryotic gene expression. In this essay, we review the basis of the current view of the regulation of elongation in the human c-myc gene and discuss similarities in elongation control among the c-myc, Drosophila hsp70 and the HIV-1 genes. Based upon these similarities, we propose a model for control of expression of these genes at the elongation phase of transcription. This model suggests that distinct promoter elements direct the assembly of RNA polymerase II transcription complexes which differ in their elongation efficiency.

In vivo footprinting of the human IL-2 gene reveals a nuclear factor bound to the transcription start site in T cells.

The IL-2 gene is a T cell specific gene that is expressed early during the activation-specific T lymphocyte development program. Electrophoretic mobility shift assay (EMSA) and DNase I footprinting assays have defined DNA/protein interactions at the IL-2 promoter cis-elements in vitro. To determine if the trans-activators documented in T cell nuclear extracts actually bind the IL-2 promoter in vivo, ligation mediated PCR (LMPCR) genomic footprinting was performed on the IL-2 promoter in both activated and non-activated T cells and HL60 promyelocytes, which do not express the IL-2 gene. The in vivo footprints indicate that the IL-2 gene transcription start site and TATA sequence are protected in both activated and resting T cells, prior to the appearance of detectable IL-2 steady state message. The distal NF-AT and the NF kappa B sites are each footprinted and the Oct/OAP site contains hypersensitive residues in the unstimulated T lymphocytes. Additional residues are protected in each of these sites after T cell activation. The proximal NF-AT site (NF-IL-2B) and the AP-1 site at -150 are protected in activated Jurkat T lymphocytes, but these two sites are not protected in activated Jurkat lymphocytes stably transfected a gene construct containing multiple NFAT binding sites.

Distinct properties of c-myc transcriptional elongation are revealed in Xenopus oocytes and mammalian cells and by template titration, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), and promoter mutagenesis.

A block to c-myc transcription elongation has been observed in Xenopus oocytes and mammalian cells. Here, we show that the distribution of RNA polymerase II transcription complexes in the c-myc promoter proximal region in Xenopus oocytes is different from that observed previously in mammalian cells. Thus, there are major differences in the c-myc elongation block observed in the two systems. In addition, as first reported for a Xenopus tubulin gene (K. M. Middleton and G. T. Morgan, Mol. Cell. Biol. 10:727-735, 1990). c-myc template titration experiments reveal the existence of two classes of RNA polymerase II transcription complexes in oocytes: one (at low template concentration) that is capable of reading through downstream sites of premature termination, and another (high template concentration) that does not. We show that these classes of polymerases are distinct from those previously identified by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), which distinguishes transcription complexes on the basis of transcribed distance, rather than on the basis of differential elongation through sites of premature termination. We also show that mutations that affect the efficiency of initiation of transcription from the c-myc P2 promoter can influence premature termination by at least two mechanisms: TATA box mutations function by the titration effect (decrease in transcription initiation results in a relative decrease in premature termination), while an upstream activator (E2F) site functions by contributing to the assembly of polymerase complexes competent to traverse the downstream sites of premature termination.

The block to transcriptional elongation within the human c-myc gene is determined in the promoter-proximal region.

A conditional block to transcriptional elongation is an important mechanism for regulating c-myc gene expression. This elongation block within the first c-myc exon was defined originally in mammalian cells by nuclear run-on transcription analyses. Subsequent oocyte injection and in vitro transcription analyses suggested that sequences near the end of the first c-myc exon are sites of attenuation and/or premature termination. We report here that the mapping of single stranded DNA in vivo with potassium permanganate (KMnO4) and nuclear run-on transcription assays reveal that polymerase is paused near position +30 relative to the major c-myc transcription initiation site. Deletion of 350 bp, including the sites of 3'-end formation and intrinsic termination defined in oocyte injection and in vitro transcription assays does not affect-the pausing of polymerase in the promoter-proximal region. In addition, sequences upstream of +47 are sufficient to confer the promoter-proximal pausing of polymerases and to generate the polarity of transcription farther downstream. Thus, the promoter-proximal pausing of RNA polymerase II complexes accounts for the block to elongation within the c-myc gene in mammalian cells. We speculate that modification of polymerase complexes at the promoter-proximal pause site may determine whether polymerases can read through intrinsic sites of termination farther downstream.

Sequences in the human c-myc P2 promoter affect the elongation and premature termination of transcripts initiated from the upstream P1 promoter.

A conditional block to transcription elongation provides one mechanism for controlling the steady-state levels of c-myc RNA in mammalian cells. Although prematurely terminated c-myc RNAs are not detectable in mammalian cells, truncated c-myc RNAs with 3' ends that map near the end of the first exon are transcribed from human c-myc templates injected into Xenopus oocytes germinal vesicles. A series of linker scanner and deletion mutants within the c-myc P2 promoter was tested in the Xenopus oocyte injection assay to determine the potential contribution of promoter elements to the elongation or premature termination of c-myc transcription. Although this analysis failed to identify sequences in the P2 promoter that significantly affect the elongation or termination of P2-initiated transcripts, our results suggest that sequences within the P2 promoter contribute to the premature termination of transcripts initiated at the upstream P1 promoter. A subset of these sequences is essential for the efficient elongation of P1-initiated transcripts through intrinsic sites of termination at the end of exon 1. These sequences affect P1 elongation when they are downstream of the site of initiation, and we hypothesize that they may be analogous to a class of prokaryotic elements required for antitermination.

Regulatory sequences of the Sgs-4 gene of Drosophila melanogaster analysed by P element-mediated transformation.

The X chromosomally located allele Sgs-4c for a larval secretion protein of Drosophila melanogaster is normally expressed in female larvae of the strain Oregon R and is hyperexpressed in male larvae exhibiting dosage compensation; the allele Sgs-4d in the strain Samarkand is weakly expressed and is not hyperexpressed in male larvae showing a dosage effect. P element-mediated transformation of upstream DNA sequences from both alleles combined with Sgs-4d coding and downstream sequences was performed to localize sequences which are responsible for the level of gene expression and for hyperexpression of Sgs-4c in male larvae. Our results demonstrate that weak expression and dosage effect are inherited with the upstream region from -1 to -838. This Samarkand fragment differs from the homologous Oregon R region only by a C to T transition at -344 which lies within an assumed binding sequence for the ecdysone receptor complex of dyad base symmetry. Replacing the Samarkand upstream region from -1 to -838 by the Oregon R region restores normal Sgs-4 expression and dosage compensation. Hyperexpression in male larvae displays high sensitivity to position effect and is nearly completely inhibited in one transformed line under heterozygous conditions. The integration of an Sgs-4d transposon into a weak spot of polytene chromosome 2L results in a decrease in gene expression. The GTT- and GT-rich regions at -1.2 and -2.0 kb do not obviously influence Sgs-4 expression but possibly play a role in induction of stage-specific chromosome puffing.

[Carcinoembryonic antigen in bronchoalveolar lavage fluids in the diagnosis of lung cancer]. / Rakovo-émbrional'nyi antigen bronkhoal'veoliarnykh smyvov v diagnostike raka legkogo.

A radioimmunoassay was used for a comparative study of CEA concentrations in the blood serum and bronchoalveolar washes-off in patients with lung cancer (27) and nontumorous lung disease (with bronchitis-14, pneumonia-18, tuberculosis-11, disseminated processes of nontumorous etiology.-12) and in persons without lung pathology (7). Diagnostic sensitivity of CEA determination in the washes-off was 89%, in the blood serum 33%; the specificity was 86 and 56.5%, respectively. The resultant diagnostic accuracy using washes-off was 86%, i.e. almost twice as high as compared to the blood serum (44.7%). Such a high accuracy in CEA determination in the bronchoalveolar washes-off provides an opportunity to use the method for differential diagnosis in unclear cases and for defining groups at high risk of developing lung cancer. The combination of a high level of CEA with unfavorable signs like basal cell hyperplasia, epidermoid metaplasia necessitate patients' follow-up.

Transformation of salivary gland secretion protein gene Sgs-4 in Drosophila: stage- and tissue-specific regulation, dosage compensation, and position effect.

The Sgs-4 gene of Drosophila melanogaster encodes one of the larval secretion proteins and is active only in salivary glands at the end of larval development. This gene lies in the X chromosome and is controlled by dosage compensation--i.e., the gene is hyperexpressed in males. Therefore, males with one X chromosome produce nearly as much Sgs-4 products as females with two X chromosomes. We used a 4.9-kilobase-pair (kb) DNA fragment containing the Sgs-4d coding region embedded in 2.6 kb of upstream sequences and 1.3 kb of downstream sequences for P-element-mediated transformation of the Sgs-4h underproducer strain Kochi-R. Sgs-4d gene expression was found in all 15 transformed lines analyzed, varying with the site of chromosomal integration. The transposed gene was subject to tissue- and stage-specific regulation. At X-chromosomal sites, the levels of gene expression were similar in both sexes, signifying dosage compensation. At autosomal sites, it was on average 1.5 times higher in males than in females. The results indicate that the transforming DNA fragment contains all sequences necessary for tissue- and stage-specific regulation and for hyperexpression in males.

Long-term exposure to sulfur dioxide, sulfuric acid mist, fly ash, and their mixtures. Results of Studies in Monkeys and guinea pigs.

Groups of cynomolgus monkeys and guinea pigs were exposed to mixtures of sulfur dioxide, fly ash, and sulfuric acid mist. The exposure concentrations varied between 0.1 and 5.0 ppm for sulfur dioxide, 0.1 and 1 mg/cu m for sulfuric acid mist, while a concentration of approximately 0.5 mg/cu m was used for fly ash. The duration of exposure was 52 weeks for guinea-pigs and 78 weeks for monkeys. Pulmonary function tests and serum biochemical and hematological analyses were conducted prior to and periodically during the exposure period. At the termination of exposure, the lungs were examined microscopically. Analysis of the data revealed that in groups exposed to the mixtures of pollutants, sulfuric acid mist was responsible for the effects observed. No synergistic action between the pollutants was detected.