Adenovirus Major Late Promoter Research Articles

Unraveling the mechanism of recognition of the 3' splice site of the adenovirus major late promoter intron by the alternative splicing factor PUF60.

Pre-mRNA splicing is critical for achieving required amounts of a transcript at a given time and for regulating production of encoded protein. A given pre-mRNA may be spliced in many ways, or not at all, giving rise to multiple gene products. Numerous splicing factors are recruited to pre-mRNA splice sites to ensure proper splicing. One such factor, the 60 kDa poly(U)-binding splicing factor (PUF60), is recruited to sites that are not always spliced, but rather function as alternative splice sites. In this study, we characterized the interaction of PUF60 with a splice site from the adenovirus major late promoter (the AdML 3' splice site, AdML3’). We found that the PUF60–AdML3’ dissociation constants are in the micromolar range, with the binding affinity predominantly provided by PUF60’s two central RNA recognition motifs (RRMs). A 1.95 Å crystal structure of the two PUF60 RRMs in complex with AdML3’ revealed a dimeric organization placing two stretches of nucleic acid tracts in opposing directionalities, which can cause looping of nucleic acid and explain how PUF60 affects pre-mRNA geometry to effect splicing. Solution characterization of this complex by light-scattering and UV/Vis spectroscopy suggested a potential 2:1 (PUF602:AdML3’) stoichiometry, consistent with the crystal structure. This work defines the sequence specificity of the alternative splicing factor PUF60 at the pre-mRNA 3’ splice site. Our observations suggest that control of pre-mRNA directionality is important in the early stage of spliceosome assembly, and advance our understanding of the molecular mechanism by which alternative and constitutive splicing factors differentiate among 3’ splice sites.

Analyzing the Dynamics of Single TBP-DNA-NC2 Complexes Using Hidden Markov Models

Single-pair Förster resonance energy transfer (spFRET) has become an important tool for investigating conformational dynamics in biological systems. To extract dynamic information from the spFRET traces measured with total internal reflection fluorescence microscopy, we extended the hidden Markov model (HMM) approach. In our extended HMM analysis, we incorporated the photon-shot noise from camera-based systems into the HMM. Thus, the variance in Förster resonance energy transfer (FRET) efficiency of the various states, which is typically a fitted parameter, is explicitly included in the analysis estimated from the number of detected photons. It is also possible to include an additional broadening of the FRET state, which would then only reflect the inherent flexibility of the dynamic biological systems. This approach is useful when comparing the dynamics of individual molecules for which the total intensities vary significantly. We used spFRET with the extended HMM analysis to investigate the dynamics of TATA-box-binding protein (TBP) on promoter DNA in the presence of negative cofactor 2 (NC2). We compared the dynamics of two promoters as well as DNAs of different length and labeling location. For the adenovirus major late promoter, four FRET states were observed; three states correspond to different conformations of the DNA in the TBP-DNA-NC2 complex and a four-state model in which the complex has shifted along the DNA. The HMM analysis revealed that the states are connected via a linear, four-well model. For the H2B promoter, more complex dynamics were observed. By clustering the FRET states detected with the HMM analysis, we could compare the general dynamics observed for the two promoter sequences. We observed that the dynamics from a stretched DNA conformation to a bent conformation for the two promoters were similar, whereas the bent conformation of the TBP-DNA-NC2 complex for the H2B promoter is approximately three times more stable than for the adenovirus major late promoter.

An Ago2-associated capped transcriptional start site small RNA suppresses adenovirus DNA replication.

Here we show that the adenovirus major late promoter produces a 31-nucleotide transcriptional start site small RNA (MLP-TSS-sRNA) that retains the 7-methylguanosine (m7G)-cap and is incorporated onto Ago2-containing RNA-induced silencing complexes (RISC) in human adenovirus-37 infected cells. RNA polymerase II CLIP (UV-cross linking immunoprecipitation) experiments suggest that the MLP-TSS-sRNA is produced by promoter proximal stalling/termination of RNA polymerase II transcription at the site of the small RNA 3′ end. The MLP-TSS-sRNA is highly stable in cells and functionally active, down-regulating complementary targets in a sequence and dose-dependent manner. The MLP-TSS-sRNA is transcribed from the opposite strand to the adenoviral DNA polymerase and preterminal protein mRNAs, two essential viral replication proteins. We show that the MLP-TSS-sRNA act in trans to reduce DNA polymerase and preterminal protein mRNA expression. As a consequence of this, the MLP-TSS-sRNA has an inhibitory effect on the efficiency of viral DNA replication. Collectively, our results suggest that this novel sRNA may serve a regulatory function controlling viral genome replication during a lytic and/or persistent adenovirus infection in its natural host.

A transcription factor IIA-binding site differentially regulates RNA polymerase II-mediated transcription in a promoter context-dependent manner

RNA polymerase II (pol II) is required for the transcription of all protein-coding genes and as such represents a major enzyme whose activity is tightly regulated. Transcriptional initiation therefore requires numerous general transcriptional factors and cofactors that associate with pol II at the core promoter to form a pre-initiation complex. Transcription factor IIA (TFIIA) is a general cofactor that binds TFIID and stabilizes the TFIID-DNA complex during transcription initiation. Previous studies showed that TFIIA can make contact with the DNA sequence upstream or downstream of the TATA box, and that the region bound by TFIIA could overlap with the elements recognized by another factor, TFIIB, at adenovirus major late core promoter. Whether core promoters contain a DNA motif recognized by TFIIA remains unknown. Here we have identified a core promoter element upstream of the TATA box that is recognized by TFIIA. A search of the human promoter database revealed that many natural promoters contain a TFIIA recognition element (IIARE). We show that the IIARE enhances TFIIA-promoter binding and enhances the activity of TATA-containing promoters, but represses or activates promoters that lack a TATA box. Chromatin immunoprecipitation assays revealed that the IIARE activates transcription by increasing the recruitment of pol II, TFIIA, TAF4, and P300 at TATA-dependent promoters. These findings extend our understanding of the role of TFIIA in transcription, and provide new insights into the regulatory mechanism of core promoter elements in gene transcription by pol II.

Effect of adenovirus infection on transgene expression under the adenoviral MLP/TPL and the CMVie promoter/enhancer in CHO cells.

The adenovirus major late promoter (MLP) and its translational regulator – the tripartite leader (TPL) sequence – can actively drive efficient gene expression during adenoviral infection. However, both elements have not been widely tested in transgene expression outside of the adenovirus genome context. In this study, we tested whether the combination of MLP and TPL would enhance transgene expression beyond that of the most widely used promoter in transgene expression in mammalian cells, the cytomegalovirus immediate early (CMVie) promoter/enhancer. The activity of these two regulatory elements was compared in Chinese hamster ovary (CHO) cells. Although transient expression was significantly higher under the control of the CMVie promoter/enhance compared to the MLP/TPL, this difference was greater at the level of transcription (30 folds) than translation (11 folds). Even with adenovirus infection to provide additional elements (in trans), CMVie promoter/enhancer exhibited significantly higher activity relative to MLP/TPL. Interestingly, the CMVie promoter/enhancer was 1.9 folds more active in adenovirus-infected cells than in non-infected cells. Our study shows that the MLP-TPL drives lower transgene expression than the CMVie promoter/enhancer particularly at the transcription level. The data also highlight the utility of the TPL sequence at the translation level and/or possible overwhelming of the cellular translational machinery by the high transcription activity of the CMVie promoter/enhancer. In addition, here we present data that show stimulation of the CMVie promoter/enhancer by adenovirus infection, which may prove interesting in future work to test the combination of CMVie/TPL sequence, and additional adenovirus elements, for transgene expression.

The adenovirus L4-22K protein regulates transcription and RNA splicing via a sequence-specific single-stranded RNA binding.

The adenovirus L4-22K protein both activates and suppresses transcription from the adenovirus major late promoter (MLP) by binding to DNA elements located downstream of the MLP transcriptional start site: the so-called DE element (positive) and the R1 region (negative). Here we show that L4-22K preferentially binds to the RNA form of the R1 region, both to the double-stranded RNA and the single-stranded RNA of the same polarity as the nascent MLP transcript. Further, L4-22K binds to a 5΄-CAAA-3΄ motif in the single-stranded RNA, which is identical to the sequence motif characterized for L4-22K DNA binding. L4-22K binding to single-stranded RNA results in an enhancement of U1 snRNA recruitment to the major late first leader 5΄ splice site. This increase in U1 snRNA binding results in a suppression of MLP transcription and a concurrent stimulation of major late first intron splicing.

639. Oncolytic Adenovirus Expressing IFN Alpha Works Synergistically with Chemoradiation

Aside from curative resection, there is no curative treatment against pancreatic adenocarcinoma (PDAC) at present. Late diagnosis and high recurrence results in five-year survival of 6%. Notably, Phase II trials based on adjuvant therapy combining systemic IFN Alpha (IFN) and chemoradiation reported 30-50% increase in two-year survival and impressive 35% increase in five-year survival of PDAC patients. Despite promising results, trial drawbacks included high patient dropout due to IFN systemic toxicity and low IFN levels in tumors. Low intratumoral IFN hampered the full potential of the therapy while IFN is known to induce tumor apoptosis, chemoradio sensitization, and decreased tumor neo-vasculatization.Aiming to improve efficacy and tolerability of IFN therapy, we have developed an oncolytic adenovirus expressing human IFN (OAd-IFN). Vector has Ad5/3 fiber modification and overexpresses Adenoviral Death Protein respectively contributing to increased infectivity and oncolysis. Taking advantage of Cox-2 up-regulation in PDAC, the Cox-2 promoter was included in the upstream of Adenovirus E1 region, restricting vector replication to cancer cells. Human IFN-alpha gene was placed in the Adenovirus E3 region in the way that its expression is controlled by the adenovirus major late promoter. Therefore, IFN expression in this vector is replication dependent. To test the vector in an immunocompetent syngeneic hamster model of pancreatic cancer, OAd-IFN expressing hamster IFN was generated. Vector contains RGD fiber modification enhancing its infectivity in hamster cells.MTS and crystal violet assays demonstrated sensitization of PDAC cells to chemotherapy (5-FU, Cisplatin, and Gemcitabine), and radiation (4 and 8Gy) by OAd-IFN. Comparison between OAd-IFN and control vector not expressing IFN (OAd-LUC) indicated that IFN expressed by OAd-IFN is functional in combination therapy sensitizing PDAC cells to chemoradiation. Colony formation assay showed that combinations of OAd-IFN with chemotherapy, radiation, or chemoradiation are synergistic and exhibit superior killing effect compared to groups without OAd-IFN.In vivo studies using immunocompetent syngeneic hamster model of pancreatic cancer showed that combinations including OAd-IFN resulted in augmented tumor shrinkage and survival compared to groups treated with chemotherapy, radiation, chemoradiation, or OAd-LUC + radiation. Hexon staining and viral DNA quantification by qPCR show OAd-IFN effectively replicates and spreads in tumors.Our data suggests OAd-IFN synergistically improves chemoradioation in PDAC cells, and shows superior therapeutic effect when treating immunocompetent model of pancreatic cancer. Vector capacity to express high levels of IFN intratumorally and the strong synergism between OAd-IFN and radiation, chemotherapy, and chemoradiation indicates OAd-IFN will contribute to more olerable and effective IFN therapy. Strong synergism between OAd-IFN and chemoradiation combined with focal expression of IFN can help to reduce not only IFN toxicity, but also dose-dependent toxicity of chemoradiation. Considering systemic IFN injection combined with chemoradiation is one of the few therapies to effectively treat pancreatic cancer, usage of OAd-IFN in combination with chemoradiation has great potential to result in more effective and tolerable therapy agaisnt pancreatic cancer.

Establishment and Validation of a Non-Radioactive Method for In Vitro Transcription Assay Using Primer Extension and Quantitative Real Time PCR.

Primer extension-dependent in vitro transcription assay is one of the most important approaches in the research field of gene transcription. However, conventional in vitro transcription assays incorporates radioactive isotopes that cause environmental and health concerns and restricts its scope of application. Here we report a novel non-radioactive method for in vitro transcription analysis by combining primer extension with quantitative real time PCR (qPCR). We show that the DNA template within the transcription system can be effectively eliminated to a very low level by our specially designed approach, and that the primers uniquely designed for primer extension and qPCR can specifically recognize the RNA transcripts. Quantitative PCR data demonstrate that the novel method has successfully been applied to in vitro transcription analyses using the adenovirus E4 and major late promoters. Furthermore, we show that the TFIIB recognition element inhibits transcription of TATA-less promoters using both conventional and nonradioactive in vitro transcription assays. Our method will benefit the laboratories that need to perform in vitro transcription but either lack of or choose to avoid radioactive facilities.

A suppressive effect of Sp1 recruitment to the first leader 5′ splice site region on L4-22K-mediated activation of the adenovirus major late promoter

Transcription from the adenovirus major late promoter (MLP) requires binding of late phase-specific factors to the so-called DE element located approximately 100 base pairs downstream of the MLP transcriptional start site. The adenovirus L4-22K protein binds to the DE element and stimulates transcription from the MLP via a DE sequence-dependent mechanism. Here we use a transient expression approach to show that L4-22K binds to an additional site downstream of the MLP start site, the so-called R1 region, which includes the major late first leader 5′ splice site. Binding of L4-22K to R1 has a suppressive effect on MLP transcription. L4-22K binds to the distal part of R1 and stimulates the recruitment of Sp1 and other cellular factors to a site overlapping the first leader 5′ splice site. Binding of Sp1 to the 5′ splice site region had an inhibitory effect on L4-22K-activated MLP transcription.

Transgene Expression under the Adenoviral Major Late Promoter, Tripartite Leader Sequence and E1 Genes in Absence and Presence of Adenovirus Infection

Active gene expression driven by the adenoviral major late promoter (MLP) and the tripartite leader sequence (TPL) influenced their utilization to drive vaccines expression. It is unclear if the complete TPL sequence is required for this active expression or if it can be further boosted in presence of the adenoviral E1 genes. In this study, we investigated the effect of TPL exones and E1 genes, out of the viral genome or under adenoviral infection, on green fluorescence protein (GFP) expression driven by the MLP. Gradual increase in the transcript copies with the addition of TPL exons indicated an additive effect especially under infection. The presence of E1 genes significantly enhanced transgene transcription. Infected cells showed higher GFP transcription and translation than non-infected cells. These results indicate higher exogenous gene expression driven by the MLP can be achieved in the presence of adenoviral components other than the TPL and E1 genes.

CTGC motifs within the HIV core promoter specify Tat-responsive pre-initiation complexes

BackgroundHIV latency is an obstacle for the eradication of HIV from infected individuals. Stable post-integration latency is controlled principally at the level of transcription. The HIV trans-activating protein, Tat, plays a key function in enhancing HIV transcriptional elongation. The HIV core promoter is specifically required for Tat-mediated trans-activation of HIV transcription. In addition, the HIV core promoter has been shown to be a potential anti-HIV drug target. Despite the pivotal role of the HIV core promoter in the control of HIV gene expression, the molecular mechanisms that couple Tat function specifically to the HIV core promoter remain unknown.ResultsUsing electrophoretic mobility shift assays (EMSAs), the TATA box and adjacent sequences of HIV essential for Tat trans-activation were shown to form specific complexes with nuclear extracts from peripheral blood mononuclear cells, as well as from HeLa cells. These complexes, termed pre-initiation complexes of HIV (PICH), were distinct in composition and DNA binding specificity from those of prototypical eukaryotic TATA box regions such as Adenovirus major late promoter (AdMLP) or the hsp70 promoter. PICH contained basal transcription factors including TATA-binding protein and TFIIA. A mutational analysis revealed that CTGC motifs flanking the HIV TATA box are required for Tat trans-activation in living cells and correct PICH formation in vitro. The binding of known core promoter binding proteins AP-4 and USF-1 was found to be dispensable for Tat function. TAR RNA prevented stable binding of PICH-2, a complex that contains the general transcription factor TFIIA, to the HIV core promoter. The impact of TAR on PICH-2 specifically required its bulge sequence that is also known to interact with Tat.ConclusionOur data reveal that CTGC DNA motifs flanking the HIV TATA box are required for correct formation of specific pre-initiation complexes in vitro and that these motifs are also required for Tat trans-activation in living cells. The impact of TAR RNA on PICH-2 stability provides a mechanistic link by which pre-initiation complex dynamics could be coupled to the formation of the nascent transcript by the elongating transcription complex. Together, these findings shed new light on the mechanisms by which the HIV core promoter specifically responds to Tat to activate HIV gene expression.

GALV expression enhances the therapeutic efficacy of an oncolytic adenovirus by inducing cell fusion and enhancing virus distribution

The limitations of the current oncolytic adenoviruses for cancer therapy include insufficient potency and poor distribution of the virus throughout the tumor mass. To address these problems, we generated an oncolytic adenovirus expressing the hyperfusogenic form of the gibbon-ape leukemia virus (GALV) envelope glycoprotein under the control of the adenovirus major late promoter. The oncolytic properties of the new fusogenic adenovirus, ICOVIR16, were analyzed both in vitro and in vivo, and compared with that of its non-fusogenic counterpart, ICOVIR15. Our results indicate that GALV expression by ICOVIR16 induced extensive syncytia formation and enhanced tumor cell killing in a variety of tumor cell types. When injected intratumorally or intravenously into mice with large pre-established melanoma or pancreatic tumors, ICOVIR16 rapidly reduced tumor burden, and in some cases, resulted in complete eradication of the tumors. Importantly, GALV expression induced tumor cell fusion in vivo and enhanced the spreading of the virus throughout the tumor. Taken together, these results indicate that GALV expression can improve the antitumoral potency of an oncolytic adenovirus and suggest that ICOVIR16 is a promising candidate for clinical evaluation in patients with cancer.

Towards rational engineering of cells: Recombinant gene expression in defined chromosomal loci

The strength of recombinant gene expression is a key property of cell lines for biopharmaceutical protein production. In most stable cell lines the expression vector is stably introduced into the host chromosomal DNA. Apart from the copy number and the used expression control elements the performance of recombinant expression vectors is modulated by genetic and epigenetic features provided by flanking host elements. Since targeted integration is very difficult cell clones with high expression of a recombinant vector are created by random integration and large scale screening for gene expression. This allows the isolation of those rare recombinant cells in which gene expression is optimal. This is usually due to locus-specific influences of the chromosomal surroundings. We have developed an efficient methodology for targeting expression cassettes to specific chromosomal sites [1,2]]. The method (Flp recombinase mediated cassette exchange - RMCE) allows the repeated use of defined loci by targeting constructs for expression of proteins and viruses, thereby allowing to exploit the positive features of a given integration site [3,4]]. Thereby, a systematic evaluation of the performance of a set of expression vectors in various chromosomal sites becomes feasible. In this study we screened for high performance integration sites in HEK293 and CHO-K1 cells supporting expression cassettes driven by a potent promoter. As a read out, production of antibodies and recombinant retroviral vectors were used. Thereby, we could show that high level expression of a given promoter is restricted to defined integration sites, while other sites show only moderate expression (data not shown). An important new finding was that a given chromosomal site is not flexible with respect to the integrated cassette but requires the integration of specific promoters. As illustrated in figure ​figure1,1, an integration site, identified for supporting high level expression of an SV40 promoter driven cassette fails to adequately support expression of MPSV and adenoviral major late promoter (AdmlP). Vice versa, another site, initially screened for supporting MSCV promoter expression, could restore expression of the highly homologous MPSV promoter while the SV40 promoter and the AdmlP promoter give only moderate expression. Figure 1 Mode of tagging defines the optimal targeting cassette. Two highly potent chromosomal integration sites were screened upon random integration of an expression cassette driven by the SV40 promoter and the MSCV promoter, respectively. By means of Flp recombinase ... Finally, we tested the impact of the orientation of the cassette in a specific chromosomal site. We found that some integration sites are flexible with respect to the orientation of the expression cassettes while others support expression only in one direction (data not shown). While classical enhancer elements are known to activate promoters largely independent from the relative position this finding suggests that other cis acting elements affect the incoming cassettes in an orientation dependent manner. Together, this shows that not the nature of integration site and the design of the vector as such define the performance of a producer cell clone. Rather, the interplay between these components defines the level and stability of expression. Since these interactions cannot be predicted, the performance of a vector in a given site has to be evaluated empirically. In order to exploit favourable sets of chromosomal sites and vectors we made use of bacterial artificial chromosome (BAC) vectors. By recombineering, expression cassettes were integrated into pre-selected chromosomal sites as encoded by BAC vectors. These vectors were randomly integrated into cells by standard transfection protocols. Clones were isolated and evaluated for expression. As expected, highly reproducible expression characteristics were found in individual clones (data not shown). In conclusion, the definition of favorable combinations of specific integration sites and vector design allow the rational exploitation of given chromosomal sites. For this purpose, technologies for site specific genetic manipulation of mammalian cells are essential. This concerns both targeted integration of expression cassettes into defined loci (such as RMCE or site specific nuclease induced homologous recombination) or by transduction of large chromosomal domains (as provided by BAC vectors). These technologies pave the way for predictable and high expression of biotechnologically relevant products such as antibodies and recombinant viral vectors.

G-actin Participates in RNA Polymerase II-dependent Transcription Elongation by Recruiting Positive Transcription Elongation Factor b (P-TEFb)

Actin is a key regulator of RNA polymerase (Pol) II-dependent transcription. Positive transcription elongation factor b (P-TEFb), a Cdk9/cyclin T1 heterodimer, has been reported to play a critical role in transcription elongation. However, the relationship between actin and P-TEFb is still not clear. In this study, actin was found to interact with Cdk9, a catalytic subunit of P-TEFb, in elongation complexes. Using immunofluorescence and immunoprecipitation assays, Cdk9 was found to bind to G-actin through the conserved Thr-186 in the T-loop. Overexpression and in vitro kinase assays showed that G-actin promotes P-TEFb-dependent phosphorylation of the Pol II C-terminal domain. An in vitro transcription experiment revealed that the interaction between G-actin and Cdk9 stimulated Pol II transcription elongation. ChIP and immobilized template assays indicated that actin recruited Cdk9 to a transcriptional template in vivo and in vitro. Using cytokine IL-6-inducible p21 gene expression system, we revealed that actin recruited Cdk9 to endogenous gene. Moreover, overexpression of actin and Cdk9 increased histone H3 acetylation and acetylized histone H3 binding to a transcriptional template through the interaction with histone acetyltransferase, p300. Taken together, our results suggested that actin participates in transcription elongation by recruiting Cdk9 for phosphorylation of the Pol II C-terminal domain, and the actin-Cdk9 interaction promotes chromatin remodeling.

Efficient and Rapid Nucleosome Traversal by RNA Polymerase II Depends on a Combination of Transcript Elongation Factors

The nucleosome is generally found to be a strong barrier to transcript elongation by RNA polymerase II (pol II) in vitro. The elongation factors TFIIF and TFIIS have been shown to cooperate in maintaining pol II in the catalytically competent state on pure DNA templates. We now show that although TFIIF or TFIIS alone is modestly stimulatory for nucleosome traversal, both factors together increase transcription through nucleosomes in a synergistic manner. We also studied the effect of TFIIF and TFIIS on transcription of nucleosomes containing a Sin mutant histone. The Sin point mutations reduce critical histone-DNA contacts near the center of the nucleosome. Significantly, we found that nucleosomes with a Sin mutant histone are traversed to the same extent and at nearly the same rate as equivalent pure DNA templates if both TFIIS and TFIIF are present. Thus, the nucleosome is not necessarily an insurmountable barrier to transcript elongation by pol II. If unfolding of template DNA from the nucleosome surface is facilitated and the tendency of pol II to retreat from barriers is countered, transcription of nucleosomal templates can be rapid and efficient.

Antibody fragments, expressed by a fowl adenovirus vector, are able to neutralize infectious bursal disease virus

Single-chain variable fragments (scFv) contain the heavy and light chain variable domains of immunoglobulin, joined by a short peptide linker. Previously, our laboratory has produced neutralizing scFv to epitopes of infectious bursal disease virus (IBDV). The in vitro delivery and expression of one of these scFv with and without the CH2-CH4 Fc domain of chicken IgY attached (scFv-Fc) by a serotype 8 fowl adenovirus (FAdV-8) vector was investigated in the present study. A panel of FAdV-8 vectors was constructed, each containing a different transgene (scFv or scFv-Fc), a different promoter to drive scFv and scFv-Fc transcription (CMVie or the fowl adenovirus major late promoter), and a different sized, right-hand end genomic deletion (52 bp or 2.3 kb). This panel was used to establish what effect these variables had on protein production, viral replication and scFv transcription, as measured by enzyme-linked imunosorbent assay and real-time polymerase chain reaction. Our results showed that, using a FAdV-8 vector containing the optimal CMVie promoter/2.3 kb deletion combination, we successfully expressed a secreted form of both scFv and scFv-Fc that were able to neutralize IBDV both in vitro and in ovo. These studies indicate that the FAdV-8 vector may be a promising candidate to deliver and express therapeutic molecules such as scFv and scFv-Fc in vivo in poultry.

Adenovirus L4-22K stimulates major late transcription by a mechanism requiring the intragenic late-specific transcription factor-binding site

The adenovirus major late promoter (MLP) generates a primary transcript that undergoes a complex pattern of regulated alternative RNA splicing and polyadenylation events. The late-specific activation of the MLP requires binding of two infected-cell specific transcription factor complexes, DEF-A and DEF-B, to the so-called DE sequence located downstream of the MLP start site. Previous studies have shown that DEF-B is a homodimer of the viral IVa2 protein and suggested that DEF-A is a heterodimer of IVa2 and an unknown protein. Two proteins from the adenoviral L4 unit have been suggested as DEF-A candidates. Here we have examined L4-22K and L4-33K for possible DEF-A activity. We show that L4-22K stimulates transcription from the MLP in a DE sequence dependent manner both in vivo and in vitro, and that L4-22K binds to the DE sequence in vitro. Further, the position of the L4-22K DNA binding site in a promoter does not appear to be critical for function. Thus, tethering L4-22K either to a position upstream or downstream of the MLP start site, or upstream of a minimal E1B promoter, resulted in an activation of transcription. We also show that the viral pIX promoter is a natural target, activated by L4-22K. Collectively, our results are compatible with the hypothesis that L4-22K may be the elusive component of DEF-A that partakes in activation of the MLP.

Proteomics Reveals a Physical and Functional Link between Hepatocyte Nuclear Factor 4α and Transcription Factor IID

Proteomic analyses have contributed substantially to our understanding of diverse cellular processes. Improvements in the sensitivity of mass spectrometry approaches are enabling more in-depth analyses of protein-protein networks and, in some cases, are providing surprising new insights into well established, longstanding problems. Here, we describe such a proteomic analysis that exploits MudPIT mass spectrometry and has led to the discovery of a physical and functional link between the orphan nuclear receptor hepatocyte nuclear factor 4alpha (HNF4alpha) and transcription factor IID (TFIID). A systematic characterization of the HNF4alpha-TFIID link revealed that the HNF4alpha DNA-binding domain binds directly to the TATA box-binding protein (TBP) and, through this interaction, can target TBP or TFIID to promoters containing HNF4alpha-binding sites in vitro. Supporting the functional significance of this interaction, an HNF4alpha mutation that blocks binding of TBP to HNF4alpha interferes with HNF4alpha transactivation activity in cells. These findings identify an unexpected role for the HNF4alpha DNA-binding domain in mediating key regulatory interactions and provide new insights into the roles of HNF4alpha and TFIID in RNA polymerase II transcription.

TAF4/4b·TAF12 Displays a Unique Mode of DNA Binding and Is Required for Core Promoter Function of a Subset of Genes

The major core promoter-binding factor in polymerase II transcription machinery is TFIID, a complex consisting of TBP, the TATA box-binding protein, and 13 to 14 TBP-associated factors (TAFs). Previously we found that the histone H2A-like TAF paralogs TAF4 and TAF4b possess DNA-binding activity. Whether TAF4/TAF4b DNA binding directs TFIID to a specific core promoter element or facilitates TFIID binding to established core promoter elements is not known. Here we analyzed the mode of TAF4b.TAF12 DNA binding and show that this complex binds DNA with high affinity. The DNA length required for optimal binding is approximately 70 bp. Although the complex displays a weak sequence preference, the nucleotide composition is less important than the length of the DNA for high affinity binding. Comparative expression profiling of wild-type and a DNA-binding mutant of TAF4 revealed common core promoter features in the down-regulated genes that include a TATA-box and an Initiator. Further examination of the PEL98 gene from this group showed diminished Initiator activity and TFIID occupancy in TAF4 DNA-binding mutant cells. These findings suggest that DNA binding by TAF4/4b-TAF12 facilitates the association of TFIID with the core promoter of a subset of genes.

Central forkhead domain of human TFIIEβ plays a primary role in binding double-stranded DNA at transcription initiation

The human general transcription factor, TFIIE, consists of two subunits, alpha and beta. Structural analyses indicated the presence of a forkhead motif within the central region of TFIIEbeta. This motif was essential for transcription and possessed a double-stranded DNA-binding activity. Protein-DNA photo-cross-linking studies indicated that TFIIEbeta binds within the promoter region, adjacent to the transcription initiation site where promoter melting begins at transcription initiation. Furthermore, neither TFIIE nor the other general transcription factor TFIIH, were required for basal transcription of adenovirus major late promoter artificially pre-melted at the initiation site. These data suggest a model in which TFIIE binds to a position adjacent to the initiation site via the forkhead domain, enabling TFIIH to begin opening the promoter. Here, we used systematic point mutations to further investigate the functional roles of this domain. The mutant proteins were expressed in bacteria, purified and used to examine transcription of two different forms of template, phosphorylation of the C-terminal domain of RNA polymerase II, as well as dsDNA-binding. Taken together, our results strongly demonstrated that the primary function of the forkhead region is dsDNA-binding in transcription. In addition, we identified three positively charged lysine residues which play a key role in this function.