Biomedical subjects

P Hearing

Publications and source records attributed to P Hearing.

At least 37 records · Page 2Linked to original sources

PubMed1993

RFX1 is identical to enhancer factor C and functions as a transactivator of the hepatitis B virus enhancer.

Hepatitis B virus gene expression is to a large extent under the control of enhancer I (EnhI). The activity of EnhI is strictly dependent on the enhancer factor C (EF-C) site, an inverted repeat that is bound by a ubiquitous nuclear protein known as EF-C. Here we report the unexpected finding that EF-C is in fact identical to RFX1, a novel transcription factor previously cloned by virtue of its affinity for the HLA class II X-box promoter element. This finding has allowed us to provide direct evidence that RFX1 (EF-C) is crucial for EnhI function in HepG2 hepatoma cells; RFX1-specific antisense oligonucleotides appear to inhibit EnhI-driven expression of the hepatitis B virus major surface antigen gene, and in transfection assays, RFX1 behaves as a potent transactivator of EnhI. Interestingly, transactivation of EnhI by RFX1 (EF-C) is not observed in cell lines that are not of liver origin, suggesting that the ubiquitous RFX1 protein cooperates with liver-specific factors.

Animals↗

PubMed1992

Different binding site requirements for binding and activation for the bipartite enhancer factor EF-1A.

The human transcription factor EF-1A binds to the purine-rich E1A core enhancer sequence in the adenovirus E1A and E4 and polyomavirus enhancer regions. The consensus binding site for EF-1A resembles that of members of the ets domain protein family. EF-1A activation of transcription requires a dimeric binding site. Analysis of binding sites containing point mutations revealed that EF-1A binding is determined by the core nucleotides of the binding site, while transcriptional activation is determined both by the core and some peripheral nucleotides that do not affect binding. We have purified EF-1A and analyzed its two constituent subunits, EF-1A alpha and EF-1A beta. EF-1A alpha (MW approximately 60kD) makes the primary DNA contacts. EF-1A beta (MW approximately 50 kD) forms a heteromultimeric complex with EF-1A alpha both in solution and on a dimeric binding site. Binding of both EF-1A subunits is necessary, but not sufficient, for transcriptional activation. We present immunochemical and functional evidence that EF-1A alpha is related to the murine ets-related protein GABP alpha and that EF-1A beta is related to the murine protein GABP beta.

Adenoviridae↗

PubMed1992

cis and trans requirements for the selective packaging of adenovirus type 5 DNA.

Polar packaging of adenovirus DNA into virions is dependent on the presence of cis-acting sequences at the left end of the viral genome. Our previous analyses demonstrated that the adenovirus type 5 (Ad5) packaging domain (nucleotides 194 to 358) is composed of at least five elements that are functionally redundant. A repeated sequence, termed the A repeat, was associated with packaging function. Here we report a more detailed analysis of the requirements for the selective packaging of Ad5 DNA. By introducing site-directed point mutations into specific A repeat sequences, we demonstrate that the A repeats represent cis-acting functional components of the packaging signal. Additional elements, located outside the originally defined packaging domain boundaries and that resemble the A repeat consensus sequence, also are capable of promoting the packaging of viral DNA. The cis-acting components of the packaging signal appear to be subject to certain spatial constraints for function, possibly reflecting a necessity for the coordinate binding of packaging proteins to these sites. In agreement with this idea, we present evidence that the interaction of a limiting trans-acting factor(s) with the packaging domain in vivo is required for efficient encapsidation of the Ad5 genome.

Adenoviruses, Human↗

PubMed1991

The C-terminal 70 amino acids of the adenovirus E4-ORF6/7 protein are essential and sufficient for E2F complex formation.

E2F is a cellular transcription factor that binds to the adenovirus (Ad) E1A enhancer and E2aE promoter regions, to the cellular c-myc P2 and dihydrofolate reductase promoters, and to other viral and cellular regulatory regions. The binding activity of E2F to the Ad E2aE promoter is dramatically increased during an adenovirus infection (termed E2F induction). E2F induction is dependent on the expression of the 150 amino acid E4-ORF6/7 protein which forms a direct, physical complex with E2F to mediate the cooperative and stable binding of E2F to inverted sites in the E2aE promoter. Using in vitro DNA binding assays to measure the formation of the infection-specific complexes, we have defined the minimal domain of the E4-ORF6/7 protein, the C-terminal 70 amino acids, required to complex with E2F and stabilize its binding at the E2aE promoter. The ability of mutant E4-ORF6/7 proteins to form the stable E2F-E2aE promoter complex in vitro correlated well with their ability to trans-activate E2 transcription in vivo. These observations support a model in which the E4-ORF6/7 protein binds to E2F to induce the cooperative binding of two E2F molecules to the E2aE promoter thereby activating E2 transcription.

Adenovirus Early Proteins↗

PubMed1991

Methylation-dependent and -independent DNA binding of nuclear factor EF-C.

Nuclear factor EF-C binds to important functional sites in the hepatitis B virus and polyomavirus enhancer regions. In this paper, we have characterized new and divergent EF-C binding sites in several viral regulatory regions. We also have demonstrated that EF-C binds to certain DNA sites only when CpG dinucleotide base pairs are methylated (m5C). EF-C binds to other sites in a methylation-independent manner. Based on similar binding properties and identical binding sites, it is very likely that EF-C corresponds to the nuclear protein MDBP previously identified by virtue of binding to methylated DNA.

Base Sequence↗

PubMed1991

Redundant elements in the adenovirus type 5 inverted terminal repeat promote bidirectional transcription in vitro and are important for virus growth in vivo.

The adenovirus inverted terminal repeat (ITR) contains a number of cis-acting elements that are involved in the initiation of viral DNA replication, as well as multiple binding motifs for the cellular transcription factors SP1 and ATF. In this study, we utilized a Hela cell transcription extract to demonstrate that the adenovirus type 5 ITR promotes bidirectional transcription in vitro. Primer extension analyses demonstrated that the ITR directed transcription at initiation sites both within the terminal repeat and at fixed distances outside of the ITR. The ITR also strongly stimulated transcription at the early region 1A (E1A) initiation site when it was situated immediately upstream of the E1A TATA box region. Deletion and point mutational analyses demonstrated that two distinct cis-acting elements were involved in these ITR-dependent transcriptional activities in vitro. Cellular transcription factors SP1 and ATF were previously shown to bind to these two regions. Analysis of viral mutants in vivo demonstrated that the NFIII/OCT-1 binding site and a conserved ATF motif were important for efficient viral growth. Regulatory elements in the ITR flanking region were found to functionally substitute for these sites.

Adenoviruses, Human↗

PubMed1991

Interaction of nuclear factor EF-1A with the polyomavirus enhancer region.

Enhancer factor 1A (EF-1A) is a mammalian nuclear protein that previously was shown to bind cooperatively to the repeated core enhancer element I sequence in the adenovirus E1A enhancer region. We now have characterized three binding sites for EF-1A in the polyomavirus A2 (Py) enhancer region. Site 1 resides in the Py A enhancer domain, and sites 2 and 3 reside in the Py B enhancer domain. EF-1A binding to Py site 1 is independent of cooperation with other EF-1A sites or the adjacent binding sites for PEA-1 and PEA-2, two murine nuclear factors that bind in the Py A enhancer domain. EF-1A binding to Py sites 2 and 3, in contrast, is cooperative, similar to the situation previously observed with binding sites in the adenovirus E1A enhancer region. In a transient replication assay, EF-1A site 1 functions synergistically with the PEA-1 and PEA-2 sites in the A enhancer domain to enhance Py replication. The functional cooperativity observed with the EF-1A, PEA-1, and PEA-2 sites in vivo does not reflect cooperative DNA binding interactions, as detected in vitro. Py EF-1A site 1 alone is capable of weakly stimulating Py replication. EF-1A site 1 overlaps with the binding sites for the murine nuclear protein PEA-3 and the ets family of oncoproteins.

Adenoviridae↗

PubMed1991

Cooperative binding of EF-1A to the E1A enhancer region mediates synergistic effects on E1A transcription during adenovirus infection.

A cellular nuclear factor, EF-1A, binds to a sequence motif which is repeated in the adenovirus type 5 E1A transcriptional control region. Previous genetic analyses demonstrated that two of these binding sites are predominant functional elements of the E1A enhancer region in vivo. In this report, we demonstrate that the cooperative binding of EF-1A to neighboring sites in the E1A enhancer region results in a synergistic activation of E1A transcription in infected cells.

Adenoviridae Infections↗

PubMed1990

Adenovirus type 5 packaging domain is composed of a repeated element that is functionally redundant.

Previous analyses have demonstrated that adenovirus DNA is packaged into virions in vivo in a polar, left-to-right fashion. The packaging of viral DNA is dependent on cis-acting elements at the left end of the genome. In this report, we describe a genetic analysis of the sequences that are required for efficient packaging of adenovirus type 5 (Ad5) DNA. Our results demonstrate that the Ad5 packaging domain (nucleotides 194 to 358) is composed of at least five distinct elements that are functionally redundant. An AT-rich repeated sequence motif, the A repeat, is located in four of five of these regions; the fifth region is also AT rich. The efficiency of viral packaging depends on the number of individual A repeats that are present in the viral genome. The deletion of the entire packaging domain resulted in the loss of virus viability. A virus that contains a multimerized oligonucleotide corresponding to A repeat II in place of the packaging domain could package viral DNA, although with reduced efficiency compared with that of the wild-type virus. Our results also suggest that the spacing of specific sequences at the left end of the Ad5 genome are important for enhancer region function in vivo.

Adenoviruses, Human↗

PubMed1989

The adenovirus early region 4 open reading frame 6/7 protein regulates the DNA binding activity of the cellular transcription factor, E2F, through a direct complex.

Nuclear factor E2F is a cellular protein that binds to the adenovirus E2 promoter and E1A enhancer regions and to the cellular c-myc P2 promoter region. The DNA binding activity of E2F, detected in vitro using nuclear extracts prepared from HeLa cells, is increased by adenovirus infection (termed E2F induction). We demonstrate here that a 19.5-kD protein, encoded by adenovirus early region 4 (E4) open reading frame (ORF) 6/7, is primarily responsible for the induction of E2F DNA binding activity to the E2 promoter region. Viral mutants that contain frame-shift mutations in E4 ORF 6/7 failed to induce E2F binding activity; a virus that carries an E4 ORF 6/7 cDNA in place of the E4-coding sequences induced E2F efficiently. Using gel mobility shift assays, we demonstrate that the E4 ORF 6/7 product induces the binding of E2F to the E2 promoter via a direct complex. The addition of a peptide-specific antiserum, directed against the E4 ORF 6/7 protein, to an in vitro E2F-binding reaction resulted in the formation of a DNA-protein complex with reduced gel mobility compared to the normal, adenovirus-induced E2F-E2 promoter complex. The formation of the E2F-E2 promoter-antibody complex was blocked by the addition of the cognate peptide used to generate the antiserum but not by a nonspecific peptide. Nuclear extracts prepared from adenovirus-infected HeLa cells were cleared of E2F binding activity using the ORF 6/7 peptide-specific serum, but not the preimmune serum, suggesting that E2F and the E4 ORF 6/7 product form a protein-protein complex in solution. The adenovirus E1A proteins are not absolutely required for the induction of E2F binding activity because the infection of HeLa cells with an E1A mutant, dl312, at high multiplicity resulted in E2F induction. Under these conditions of infection, the E4 ORF 6/7 product was synthesized. E2F binding activity was induced, but inefficiently, in cells infected with E4 ORF 6/7 mutants, indicating that an additional pathway may lead to E2F induction.

Adenoviridae↗

PubMed1989

Adenovirus early region 4 encodes two gene products with redundant effects in lytic infection.

In order to assign specific functions to individual gene products encoded by adenovirus type 5 early region 4 (E4), we have constructed and analyzed a set of mutant viruses that express individual E4 open reading frames or combinations of open reading frames. The results of these analyses demonstrate that the gene products of E4 open reading frames 3 and 6 have redundant effects in viral lytic infection. These E4 products independently augment viral DNA replication, viral late protein synthesis, the shutoff of host cell protein synthesis, and the production of infectious virus. The product of open reading frame 6 is more efficient in the regulation of these processes than is the product of open reading frame 3. The regulation of viral DNA replication and the control of viral and cellular protein synthesis appear to be separable functions associated with both E4 gene products. The role of early region 4 in adeno-associated virus helper function, however, is mediated only by the product of open reading frame 6. Finally, we demonstrate that E4 mutant viruses display a multiplicity-leakiness phenotype which is consistent with the regulatory role that this region plays in viral infection.

Adenoviruses, Human↗

PubMed1989

Independent cyclic AMP and E1A induction of adenovirus early region 4 expression.

A cellular transcription factor, ATF, binds to a repeated element in the adenovirus early region 4 (E4) promoter. ATF also binds to other viral early promoter regions and to the cyclic AMP (cAMP) response elements of cellular genes. In this report, we demonstrate that a single ATF-binding site located immediately upstream of the E4 TATA box, between -62 and -46, mediates induction of E4 transcription by 8-bromoadenosine-3',5-cyclic monophosphate or cholera toxin in the human hepatoma cell line HepG2 and rat pheochromocytoma cell line PC12. Different ATF-binding sites in the E4 control region independently conferred cAMP inducibility on the simian virus 40 early promoter in PC12 cells. Induction of E4 expression by cAMP was also observed in virus-infected HepG2 cells. Other viral early promoter regions that contain ATF-binding sites (E1A and E2A) were also induced by cAMP in infected cells. E4 expression was activated by the E1A 13S mRNA products in HepG2 cells. E1A trans activation appears to be distinct from the cAMP response.

Adenovirus Early Proteins↗

PubMed1989

Nuclear factor EF-1A binds to the adenovirus E1A core enhancer element and to other transcriptional control regions.

We have identified a cellular enhancer-binding protein, present in nuclear extracts prepared from human and rodent cells, that binds to the adenovirus E1A enhancer element I sequence. The factor has been termed EF-1A, for enhancer-binding factor to the E1A core motif. EF-1A was found to bind to two adjacent, related sequence motifs in the E1A enhancer region (termed sites A and B). The binding of EF-1A to these adjacent sites, or to synthetic dimerized sites of either motif, was cooperative. The cooperative binding of EF-1A to these sites was not subject to strict spacing constraints. EF-1A also bound to related sequences upstream of the E1A enhancer region and in the polyomavirus and adenovirus E4 enhancer regions. The EF-1A-binding region in the E1A enhancer stimulated expression of a linked gene in human 293 cells when multimerized. Based on the contact sites for EF-1A binding determined by chemical interference assays, this protein appears to be distinct from any previously characterized nuclear binding protein.

Adenoviridae↗

PubMed1989

Binding of nuclear factor EF-C to a functional domain of the hepatitis B virus enhancer region.

Nuclear factor EF-C is present in extracts prepared from human HepG2 liver cells and from other, nonliver cell lines and binds to the hepatitis B virus and polyomavirus transcriptional enhancer regions in vitro. An inverted repeat (5'-GTTGCNNNGCAAC-3') is located within both binding regions. Diethyl pyrocarbonate interference binding assays and competition binding experiments using altered binding sites demonstrated that EF-C contacts symmetrical nucleotides within the inverted repeat. Mutations that changed the length of the spacer region between the arms of the inverted repeat were introduced in the hepatitis enhancer region. Introduction of 1 or 2 base pairs between the repeats did not affect EF-C binding, but deletion of 1 base pair or introduction of 3 to 9 base pairs reduced binding dramatically. Introduction of 10 base pairs restored partial EF-C binding ability. These and other results suggest that EF-C binding is stabilized by dimerization. In vivo assays for enhancer function using these mutants demonstrated that the EF-C binding site is a functional and important component of the hepatitis B virus enhancer region.

Base Composition↗

PubMed1988

EivF, a factor required for transcription of the adenovirus EIV promoter, binds to an element involved in EIa-dependent activation and cAMP induction.

The isolation of a cellular factor that was specifically required for transcription from the promoter of the adenovirus early gene iv, an EIa-activated promoter, is described. This factor (EivF) was purified from HeLa cells using a functional transcription assay and identified as a 72,000- to 65,000-dalton protein. DNase I footprinting experiments demonstrated that purified EivF bound to the sequence 5'-GT(G/T)ACGT-3' present two times upstream of the Eiv TATA box. Nuclear extracts prepared from HeLa cells contained more than one factor capable of binding to the EivF recognition site. Previous studies have indicated that a sequence similar to the EivF-binding site was recognized by a 43,000-dalton protein and participated in the cAMP response of the somatostatin promoter. The purified and transcriptionally active EivF also bound to DNA sequence elements present in the somatostatin and alpha-gonadotropin promoters shown previously to be responsive to cAMP.

Adenoviridae↗

PubMed1988

Cellular transcription factor binds to adenovirus early region promoters and to a cyclic AMP response element.

We have analyzed the sequences that regulate the transcription of adenovirus type 5 early region 4 (E4). A region located immediately upstream of the E4 TATA box is required for efficient E4 transcription in vitro and in vivo. A cellular transcription factor, termed ETF-A, binds to this region. ETF-A also binds to additional sites in the E4 5'-flanking region, including the adenovirus terminal repeat, as well as to the adenovirus early region 2 promoter region and the adeno-associated virus early promoter region. A repeated sequence motif is found in each binding domain. The same factor binds to a region upstream of a cellular gene that contains a cyclic AMP response element. Consistent with this result, E4 expression is induced in vivo by cyclic AMP. Two other regions further upstream of the E4 initiation site also contribute to efficient E4 expression. These domains are functionally redundant and contain binding sites for ETF-A. One domain is the adenovirus terminal repeat, which has strong promoter activity in vitro and in vivo.

Adenoviruses, Human↗

PubMed1988

Multiple negative elements upstream of the murine c-myc gene share nuclear factor binding sites with SV40 and polyoma enhancers.

We previously identified a 716 bp DNA segment, 424 to 1140 bp 5' of the murine c-myc gene, which exhibited the properties of a transcriptional 'dehancer' because it negated the effects of the SV40 enhancer (Remmers et al., 1986). Here, we show that this 716 bp 'dehancer region' is composed of multiple negative elements each of which functions at a distance of > 1 Kb to inhibit the SV40 enhancer. One of these negative elements displays lymphoid cell specificity. HeLa cell nuclear factor binding sites are found within these negative effector sequences. One of these binding sites is related to that of the Py EF-C transcription factor which recognizes a short dyad symmetry element within the polyoma enhancer. Interestingly, a dimer of the Py EF-C recognition sequence exhibited a strong 'dehancer effect' in the pSV2CAT expression vector. These negative elements may function by interfering with transcription factors required for SV40 enhancer activity.

Animals↗

PubMed1987

Analysis of adenovirus early region 4-encoded polypeptides synthesized in productively infected cells.

Peptide-specific antisera were developed to analyze the products encoded by adenovirus type 5 early region 4 (E4) open reading frames 6 and 7. Reading frame 6 previously was shown to encode a 34-kilodalton polypeptide (34K polypeptide) that forms a complex with the early region 1B (E1B)-55K antigen and is required for efficient viral growth in lytic infection. Antisera that were generated recognized the E4-34K protein as well as a family of related polypeptides generated by the fusion of open reading frames 6 and 7. These polypeptides shared amino-terminal sequences with the 34K protein. Short-pulse analysis suggested that the heterogeneity observed with the 6/7 fusion products resulted from differential splicing patterns of related E4 mRNAs. An antiserum directed against the amino terminus of reading frame 6 recognized only the free form of the 34K antigen that was not associated with the E1B-55K protein. This observation allowed the determination of the stability of the free and complexed form of this polypeptide. Pulse-chase analyses demonstrated that both forms of the 34K protein had half-lives greater than 24 h, suggesting that complex formation did not result in stabilization of this gene product. The half-lives of the 6/7 fusion products were approximately 4 h. The 34K protein also was shown to have a nuclear localization within infected cells. Finally, analysis of a mutant carrying deletions in both the E4-34K and E1B-55K polypeptides indicated that the complex formed between these two proteins was a functional unit in lytic infection.

Adenovirus Early Proteins↗