NORMAL GENOMIC

• Jonker HR, Wechselberger RW, Boelens R, Kaptein R, Folkers GE
• The intrinsically unstructured domain of PC4 modulates the activity of the structured core through inter- and intramolecular interactions.
• Biochemistry. 2006; 45: 5067-81
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• Proteins frequently contain unstructured regions apart from a functionally important and well-conserved structured domain. Functional and structural aspects for these regions are frequently less clear. The general human positive cofactor 4 (PC4), has such a domain organization and can interact with various DNA substrates, transcriptional activators, and basal transcription factors. While essential for the cofactor function, structural and functional knowledge about these interactions is limited. Using biochemical, nuclear magnetic resonance (NMR), and docking experiments, we show that the carboxy-terminal structured core domain (PC4ctd) is required and sufficient for binding to single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and the herpes simplex virion protein 16 (VP16) activation domain (VP16ad). We determined the interaction surfaces within PC4 and showed that VP16 and DNA binding are mutually exclusive. Although the amino-terminal domain of PC4 (PC4ntd) alone is devoid of any bioactivity, it increases the interaction with VP16ad. While it decreases the ssDNA-binding and DNA-unwinding activity, it does not influence dsDNA binding. Structural characterization of this domain showed that it is highly flexible and mostly unstructured both in the free form and in the complex. NMR titration experiments using various protein and DNA substrates of the individual domains and the full-length PC4 revealed local conformational or environmental changes in both the structured and unstructured subdomains, which are interpreted to be caused by inter- and intramolecular interactions. We propose that the unstructured PC4ntd regulates the PC4 cofactor function by specific interactions with the activator and through modulation and/or shielding of the interaction surface in the structured core of PC4ctd.

• Ottosen S et al.
• Phosphorylation of the VP16 transcriptional activator protein during herpes simplex virus infection and mutational analysis of putative phosphorylation sites.
• Virology. 2006; 345: 468-81
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• VP16 is a virion phosphoprotein of herpes simplex virus and a transcriptional activator of the viral immediate-early (IE) genes. We identified four novel VP16 phosphorylation sites (Ser18, Ser353, Ser411, and Ser452) at late times in infection but found no evidence of phosphorylation of Ser375, a residue reportedly phosphorylated when VP16 is expressed from a transfected plasmid. A virus carrying a Ser375Ala mutation of VP16 was viable in cell culture but with a slow growth rate. The association of the mutant VP16 protein with IE gene promoters and subsequent IE gene expression was markedly reduced during infection, consistent with prior transfection and in vitro results. Surprisingly, the association of Oct-1 with IE promoters was also diminished during infection by the mutant strain. We propose that Ser375 is important for the interaction of VP16 with Oct-1, and that the interaction is required to enable both proteins to bind to IE promoters.

• Yatsula B et al.
• Identification of binding sites of EVI1 in mammalian cells.
• J Biol Chem. 2005; 280: 30712-22
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• The leukemia-associated protein EVI1 possesses seven zinc fingers within an N-terminal domain (amino acids 1-250) that binds to GACAAGATA. Single amino acid missense mutants of EVI1 were developed that failed to bind DNA either in vitro, as assessed by gel shift assay, or in vivo, as shown by transactivation studies. Specifically, mutation R205N lacks high affinity binding to the GACAAGATA motif. Putative EVI1 target genes were identified by using an EVI1-(1-250)-VP16 fusion protein that acts as a transcriptional activator with the binding specificity of EVI1. Sixteen genes induced in NIH 3T3 cells by wild type EVI1-VP16 but not by mutant forms were identified. Sequence analysis revealed evolutionarily conserved GACAAGATA-like motifs within 10 kb of their transcription start sites, and by chromatin immunoprecipitation in fibroblasts, we showed occupancy of many of these sites by EVI1-VP16. To assess whether native EVI1 binds to these sites in EVI1-transformed myeloid cells, we performed chromatin immunoprecipitation in 32Dcl3 and NFS58 cells, using anti-EVI1 antisera, and we showed that the majority of these sites is bound by wild type EVI1. These putative target genes include Gadd45g, Gata2, Zfpm2/Fog2, Skil (SnoN), Klf5 (BTEB2), Dcn, and Map3k14 (Nik). In this study we demonstrated for the first time that the N-terminal DNA binding domain of EVI1 has the capacity to bind to endogenous genes. We hypothesized that these genes play a critical role in EVI1-induced transformation.

• Martin SE, Mu X, Klein WH
• Identification of an N-terminal transcriptional activation domain within Brn3b/POU4f2.
• Differentiation. 2005; 73: 18-27
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• The POU-domain transcription factor Brn3b/ POU4f2 is an essential regulator of gene expression in mouse retinal ganglion cells. Although Brn3b's importance in the differentiation of these cells has been firmly established, the regions on Brn3b where transcriptional activation and/or repression domains reside are only vaguely defined, and conflicting publications report both activation and repression activities for Brn3b. To clarify its function, we monitored the transcriptional activity of Brn3b and Gal4 DNA-binding domain (DBD)-Brn3b fusion proteins in cotransfection experiments using either Brn3-consensus or Gal4 DNA-binding sites to drive reporter gene expression. At Gal4 DNA-binding sites, transrepression activity mapping to the POU domain within Brn3b's C-terminal region masked any transactivation activity. More detailed experiments revealed that expressing abnormally high levels of POU homeodomain- or other homeodomain-containing sequences caused fortuitous transrepression in the cotransfection assay. To avoid transrepression, Brn3b sequences lacking Brn3b's POU domain were fused to the Gal4 DBD to allow identification of regions that were responsible for transcriptional activation. Considerable transactivation activity was located between amino acid residues 100 and 239, although other regions also had activity. The transactivation domain synergized strongly with another transcription factor, LexA-VP16. At Brn3 DNA-binding sites, full-length Brn3b increased transcription more than 25-fold, and similar activation was observed with the closely related factor Brn3a/POU4f1. No transactivation activity was associated with the C-terminal POU domain-containing portion of Brn3b. The results demonstrate that Brn3b regulates gene expression through the action of a strong transcriptional activation domain within its N-terminal sequence.

• Ferreira ME, Hermann S, Prochasson P, Workman JL, Berndt KD, Wright AP
• Mechanism of transcription factor recruitment by acidic activators.
• J Biol Chem. 2005; 280: 21779-84
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• Many transcriptional activators are intrinsically unstructured yet display unique, defined conformations when bound to target proteins. Target-induced folding provides a mechanism by which activators could form specific interactions with an array of structurally unrelated target proteins. Evidence for such a binding mechanism has been reported previously in the context of the interaction between the cancer-related c-Myc protein and the TATA-binding protein, which can be modeled as a two-step process in which a rapidly forming, low affinity complex slowly converts to a more stable form, consistent with a coupled binding and folding reaction. To test the generality of the target-induced folding model, we investigated the binding of two widely studied acidic activators, Gal4 and VP16, to a set of target proteins, including TATA-binding protein and the Swi1 and Snf5 subunits of the Swi/Snf chromatin remodeling complex. Using surface plasmon resonance, we show that these activator-target combinations also display bi-phasic kinetics suggesting two distinct steps. A fast initial binding phase that is inhibited by high ionic strength is followed by a slow phase that is favored by increased temperature. In all cases, overall affinity increases with temperature and, in most cases, with increased ionic strength. These results are consistent with a general mechanism for recruitment of transcriptional components to promoters by naturally occurring acidic activators, by which the initial contact is mediated predominantly through electrostatic interactions, whereas subsequent target-induced folding of the activator results in a stable complex.

• Misra V, Rapin N, Akhova O, Bainbridge M, Korchinski P
• Zhangfei is a potent and specific inhibitor of the host cell factor-binding transcription factor Luman.
• J Biol Chem. 2005; 280: 15257-66
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• Host cell factor (HCF) was initially discovered as a cellular co-factor required for the activation of herpes simplex virus immediate early gene expression by the virion associated transactivator VP16. HCF also participates in a variety of cellular processes, although the mechanism of its action is not known. VP16 binds to HCF through a 4-amino acid motif (EHAY), which closely resembles the HCF binding domain of two cellular basic leucine-zipper proteins, Luman and Zhangfei. Luman is a powerful transcription factor that, in transient expression assays, activates promoters containing cAMP or unfolded protein response elements (UPRE). In contrast, Zhangfei neither binds consensus recognition elements for basic leucine-zipper proteins nor does it activate promoters containing them. Here we show that Zhangfei suppresses the ability of Luman to activate transcription. HCF appeared to be required for efficient suppression. A mutant of Zhangfei, which was unable to bind HCF, was impaired in its ability to suppress Luman. Zhangfei did not suppress ATF6, a transcription factor closely related to Luman but that does not bind HCF, unless the HCF binding motif of Luman was grafted onto it. Zhangfei inhibited the HCF-dependent activation of a UPRE-containing promoter by a Gal4-Luman fusion protein but was unable to inhibit the HCF-independent activation by Gal4-Luman of a promoter that contained Gal4 binding motifs. Binding of HCF by Zhangfei was required for the co-localization of Luman and Zhangfei to nuclear domains, suggesting that HCF might target the proteins to a common location.

• Robinson KM, Schultz MC
• Gal4-VP16 directs ATP-independent chromatin reorganization in a yeast chromatin assembly system.
• Biochemistry. 2005; 44: 4551-61
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• Major insights into the regulation of chromatin organization have stemmed from biochemical studies using Gal4-VP16, a chimeric transcriptional activator in which the DNA binding domain of Gal4p is fused to the activation domain of viral protein VP16. Unexpectedly, given previous intensive efforts to understand how Gal4-VP16 functions in the context of chromatin, we have uncovered a new mode of chromatin reorganization that is dependent on Gal4-VP16. This reorganization is performed by an activity in a crude DEAE (CD) fraction from budding yeast which also supports ATP-dependent assembly of physiologically spaced nucleosome arrays. Biochemical analysis reveals that the activity tightly associates with chromatin and reorganizes nucleosome arrays by a mechanism which is insensitive to ATP depletion after nucleosome assembly. It generates a chromatin organization in which a nucleosome is stably positioned immediately adjacent to Gal4p binding sites in the template DNA. Individual deletion of genes previously implicated in chromatin assembly and remodeling, namely, the histone chaperones NAP1, ASF1, and CAC1 and the SNF2-like DEAD/H ATPases SNF2, ISW1, ISW2, CHD1, SWR1, YFR038w, and SPT20, does not significantly perturb reorganization. Therefore, Gal4-VP16-directed chromatin reorganization in yeast can occur by an ATP-independent mechanism that does not require SAGA, SWI/SNF, Isw1, or Isw2 chromatin remodeling complexes.

• Yang F, DeBeaumont R, Zhou S, Naar AM
• The activator-recruited cofactor/Mediator coactivator subunit ARC92 is a functionally important target of the VP16 transcriptional activator.
• Proc Natl Acad Sci U S A. 2004; 101: 2339-44
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• The human activator-recruited cofactor (ARC), a family of large transcriptional coactivator complexes related to the yeast Mediator, was recently identified based on functional association with the activation domains of multiple cellular and viral transcriptional activators, including the herpes simplex viral activator VP16, sterol regulatory element binding protein, and NF-kappaB. Here we describe the biochemical purification and cloning of the 92-kDa ARC/Mediator subunit, ARC92, that is specifically targeted by the activation domain of the VP16 transactivator. Affinity chromatography using the VP16 activation domain followed by peptide microsequencing led to the identification of ARC92 as a specific cellular interaction partner of the VP16 activation domain. ARC92 associates with the VP16 activation domain in vitro and in vivo, and the VP16 binding domain of ARC92 is a strong competitive inhibitor of Gal4-VP16 in vivo. Moreover, small interfering RNA-mediated knockdown of ARC92 in human cells results in selective inhibition of Gal4-VP16 gene activation. Taken together, our results suggest that ARC92 is a direct and specific target of the VP16 transactivator that serves in the context of the ARC/Mediator coactivator as an important transducer of transcription activating signals from the VP16 activation domain to the RNA polymerase II transcriptional machinery.

• Schmaus S, Wolf H, Schwarzmann F
• The reading frame BPLF1 of Epstein-Barr virus: a homologue of herpes simplex virus protein VP16.
• Virus Genes. 2004; 29: 267-77
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• The open reading frame BPLF1 of Epstein-Barr virus (EBV) shows homology to the Herpes simplex virus 1 (HSV1) protein VP16. This protein is a structural tegument component playing a pivotal role for HSV replication as trans-activator of viral immediate-early genes. An EBV gene with a comparable function has not been described so far. However, computer analysis indicated that BPLF1 may be a tegument protein homologous to VP16. This is the first report on the characterisation of the BPLF1 gene, its transcription, and expression of its gene product in vitro and in vivo. Using RT-PCR and Northern blot assays we demonstrated that the BPLF1 gene belongs to the class of late lytic cycle genes of EBV. Besides a full length transcript of 9.5 kb also a polyadenylated transcript of approximately 3 kb is synthesised. However, no consensus splice sites could be identified. Northern blot experiments using partially overlapping probes and sequencing of a BPLF1-specific cDNA revealed 1,550 nucleotides of the BPLF1 transcript, collinear in sequence with the viral genome from position 64547 to 66097. A recombinant Western blot assay detected BPLF1-specific antibodies in seropositive individuals, in particular in cases with elevated viral replication like infectious mononucleosis, chronic active infection, and nasopharyngeal carcinoma. This demonstrated expression of the BPLF1 protein in vivo. Thus, experimental data and computer analysis strongly support the hypothesis of BPLF1 being a tegument protein of the EBV homologous to VP16 of HSV1 and ORF22 of Varicella zoster virus.

• Herrera FJ, Triezenberg SJ
• VP16-dependent association of chromatin-modifying coactivators and underrepresentation of histones at immediate-early gene promoters during herpes simplex virus infection.
• J Virol. 2004; 78: 9689-96
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• During infection by herpes simplex virus type 1 (HSV-1), the virion protein VP16 activates the transcription of viral immediate-early (IE) genes. Genetic and biochemical assays have shown that the potent transcriptional activation domain of VP16 can associate with general transcription factors and with chromatin-modifying coactivator proteins of several types. The latter interactions are particularly intriguing because previous reports indicate that HSV-1 DNA does not become nucleosomal during lytic infection. In the present work, chemical cross-linking and immunoprecipitation assays were used to probe the presence of activators, general transcription factors, and chromatin-modifying coactivators at IE gene promoters during infection of HeLa cells by wild-type HSV-1 and by RP5, a viral strain lacking the VP16 transcriptional activation domain. The presence of VP16 and Oct-1 at IE promoters did not depend on the activation domain. In contrast, association of RNA polymerase II, TATA-binding protein, histone acetyltransferases (p300 and CBP), and ATP-dependent remodeling proteins (BRG1 and hBRM) with IE gene promoters was observed in wild-type infections but was absent or reduced in cells infected by RP5. In contrast to the previous evidence for nonnucleosomal HSV-1 DNA, histone H3 was found associated with viral DNA at early times of infection. Interestingly, histone H3 was underrepresented on IE promoters in a manner dependent on the VP16 activation domain. Thus, the VP16 activation domain is responsible for recruiting general transcription factors and coactivators to IE promoters and also for dramatically reducing the association of histones with those promoters.

• Nedialkov YA, Triezenberg SJ
• Quantitative assessment of in vitro interactions implicates TATA-binding protein as a target of the VP16C transcriptional activation region.
• Arch Biochem Biophys. 2004; 425: 77-86
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• Models of mechanisms of transcriptional activation in eukaryotes frequently invoke direct interactions of transcriptional activation domains with target proteins including general transcription factors or coactivators such as chromatin modifying complexes. The potent transcriptional activation domain (AD) of the VP16 protein of herpes simplex virus has previously been shown to interact with several general transcription factors including the TATA-binding protein (TBP), TBP-associated factor 9 (TAF9), TFIIA, and TFIIB. In surface plasmon resonance assays, a module of the VP16 AD designated VP16C (residues 452-490) bound to TBP with an affinity notably stronger than to TAF9, TFIIA or TFIIB. Moreover, the interaction of VP16C with TBP correlated well with transcriptional activity for a panel of VP16C substitution variants. These results support models in which the interactions of ADs with TBP play an important role in transcriptional activation.

• Knez J, Bilan PT, Capone JP
• A single amino acid substitution in herpes simplex virus type 1 VP16 inhibits binding to the virion host shutoff protein and is incompatible with virus growth.
• J Virol. 2003; 77: 2892-902
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• In addition to its well-established role in the activation of herpes simplex virus immediate-early gene transcription, VP16 interacts with and downregulates the function of the virion host shutoff protein (vhs), thereby attenuating vhs-mediated destruction of viral mRNAs and translational arrest at late times of infection. We have carried out two-hybrid analysis in vivo and protein-protein interaction assays in vitro to identify determinants in VP16 necessary for interaction with vhs. The minimal amino-terminal subfragment of VP16 capable of binding to vhs encompassed residues 1 to 345. Alteration of a single leucine at position 344 to alanine (L344A) in the context of the amino-terminal fragment of VP16 containing residues 1 to 404 was sufficient to abolish interaction with vhs in vitro and in vivo. Leu344 could be replaced with hydrophobic amino acids (Ile, Phe, Met, or Val) but not by Asn, Lys, or Pro, indicating that hydrophobicity is an important property of binding to vhs. VP16 harboring a loss-of-function mutation at L344 was not compromised in its ability to interact with host cell factor (HCF-1) or to activate transcription of viral immediate-early genes in transient-transfection assays. Virus complementation assays using the VP16-null virus 8MA and the VP16/vhs double-mutant virus 8MAdeltaSma showed that VP16(L344A) was able to complement the growth of 8MAdeltaSma but not 8MA. Thus, a single point mutation in VP16 uncouples binding to vhs from other functions of VP16 required for virus growth and indicates that direct physical association between VP16 and vhs is necessary to sustain a productive infection.

• Luciano RL, Wilson AC
• An activation domain in the C-terminal subunit of HCF-1 is important for transactivation by VP16 and LZIP.
• Proc Natl Acad Sci U S A. 2002; 99: 13403-8
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• In herpes simplex virus, lytic replication is initiated by the viral transactivator VP16 acting with cellular cofactors Oct-1 and HCF-1. Although this activator complex has been studied in detail, the role of HCF-1 remains elusive. Here, we show that HCF-1 contains an activation domain (HCF-1(AD)) required for maximal transactivation by VP16 and its cellular counterpart LZIP. Expression of the VP16 cofactor p300 augments HCF-1(AD) activity, suggesting a mechanism of synergy. Infection of cells lacking the HCF-1(AD) leads to reduced viral immediate-early gene expression and lowered viral titers. These findings underscore the importance of HCF-1 to herpes simplex virus replication and VP16 transactivation.

• Hall DB, Struhl K
• The VP16 activation domain interacts with multiple transcriptional components as determined by protein-protein cross-linking in vivo.
• J Biol Chem. 2002; 277: 46043-50
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• Transcriptional activator proteins recruit the RNA polymerase II machinery and chromatin-modifying activities to promoters. Biochemical experiments indicate that activator proteins can associate with a large number of proteins, and many such proteins have been proposed to be direct targets of activators. However, there is great uncertainty about which biochemical interactions are physiologically relevant. Here, we develop a formaldehyde-based cross-linking procedure to identify protein-protein interactions that occur under physiological conditions. We show that the VP16 activation domain directly interacts with TATA-binding protein (TBP), TFIIB, and the SAGA histone acetylase complex in vivo.

• Grossmann JG, Sharff AJ, O'Hare P, Luisi B
• Molecular shapes of transcription factors TFIIB and VP16 in solution: implications for recognition.
• Biochemistry. 2001; 40: 6267-74
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• The molecular shapes of transcription factors TFIIB and VP16 have been studied by small-angle X-ray scattering (SAXS). We interpret the shapes and discuss the implications for the specific recruitment of these proteins into regulatory assemblies. Human transcription factor TFIIB, a universal component of the transcription preinitiation complex, has a triangular form resulting from intramolecular associations between its two principal structural domains. A segment linking the two domains appears to be conformationally flexible. The solution shape of TFIIB can be well fitted with the crystal structure of the DNA-bound C-terminal domain together with the NMR structure of the N-terminal domain; however, the shape cannot accommodate the NMR structure of the isolated C-terminal domain. We discuss how the conformational differences between the solution structures of the isolated C-terminal domain and the intact protein might result from interdomain allostery. Docking the SAXS shape of intact TFIIB into the preinitiation complex suggests that the flexible linker region may contact the 3' flanking region of the TATA element in the major groove. Transcription rates can be enhanced by activator proteins, and the classical example is the herpes simplex virus factor VP16 (alpha-TIF), which associates with cellular transcription factors, including TFIIB. The shape reconstruction of VP16 from its SAXS profile reveals a globular structural core that can be well modeled by the crystal structure of a conserved, central region of the protein. However, the carboxy terminus extends from this core and is essentially disordered. As it makes defined protein-protein interactions in the activation complex, the flexible segment is likely to condense upon assembly with its partners.

• Lee S, Herr W
• Stabilization but not the transcriptional activity of herpes simplex virus VP16-induced complexes is evolutionarily conserved among HCF family members.
• J Virol. 2001; 75: 12402-11
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• The human herpes simplex virus (HSV) protein VP16 induces formation of a transcriptional regulatory complex with two cellular factors-the POU homeodomain transcription factor Oct-1 and the cell proliferation factor HCF-1-to activate viral immediate-early-gene transcription. Although the cellular role of Oct-1 in transcription is relatively well understood, the cellular role of HCF-1 in cell proliferation is enigmatic. HCF-1 and the related protein HCF-2 form an HCF protein family in humans that is related to a Caenorhabditis elegans homolog called CeHCF. In this study, we show that all three proteins can promote VP16-induced-complex formation, indicating that VP16 targets a highly conserved function of HCF proteins. The resulting VP16-induced complexes, however, display different transcriptional activities. In contrast to HCF-1 and CeHCF, HCF-2 fails to support VP16 activation of transcription effectively. These results suggest that, along with HCF-1, HCF-2 could have a role, albeit probably a different role, in HSV infection. CeHCF can mimic HCF-1 for both association with viral and cellular proteins and transcriptional activation, suggesting that the function(s) of HCF-1 targeted by VP16 has been highly conserved throughout metazoan evolution.

• Babb R, Huang CC, Aufiero DJ, Herr W
• DNA recognition by the herpes simplex virus transactivator VP16: a novel DNA-binding structure.
• Mol Cell Biol. 2001; 21: 4700-12
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• Upon infection, the herpes simplex virus (HSV) transcriptional activator VP16 directs the formation of a multiprotein-DNA complex-the VP16-induced complex-with two cellular proteins, the host cell factor HCF-1 and the POU domain transcription factor Oct-1, on TAATGARAT-containing sequences found in the promoters of HSV immediate-early genes. HSV VP16 contains carboxy-terminal sequences important for transcriptional activation and a central conserved core that is important for VP16-induced complex assembly. On its own, VP16 displays little, if any, sequence-specific DNA-binding activity. We show here that, within the VP16-induced complex, however, the VP16 core has an important role in DNA binding. Mutation of basic residues on the surface of the VP16 core reveals a novel DNA-binding surface with essential residues which are conserved among VP16 orthologs. These results illuminate how, through association with DNA, VP16 is able to interpret cis-regulatory signals in the DNA to direct the assembly of a multiprotein-DNA transcriptional regulatory complex.

• Lu R, Misra V
• Zhangfei: a second cellular protein interacts with herpes simplex virus accessory factor HCF in a manner similar to Luman and VP16.
• Nucleic Acids Res. 2000; 28: 2446-54
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• Host cell factor (HCF, C1, VCAF or CFF) is a cellular protein that is required for transcription activation of herpes simplex virus (HSV) immediate-early (IE) genes by the virion protein VP16. The biological function of HCF remains unclear. Recently we identified a cellular transcription activator, Luman. As with VP16, the transactivation function of Luman is also regulated by HCF. Here we report a second human protein, Zhangfei (ZF) that interacts with HCF in a fashion similar to Luman and VP16. Although ZF shares no significant sequence homology with Luman, the two proteins have some structural similarities. These include: a basic domain-leucine zipper (bZIP) region, an acidic activation domain and a consensus HCF-binding motif. Unlike Luman, or most other bZIP proteins, ZF by itself did not appear to bind consensus bZIP-binding sites. It was also unable to activate promoters containing these response elements. Although in transient expression assays ectopically expressed ZF was unable to block transactivation by VP16 of a HSV IE promoter, ZF could prevent the expression of several HSV proteins in cells infected with the virus. The ability of ZF to block the synthesis of the HSV IE protein ICP0 relied on its binding to HCF, since a mutant of ZF that was unable to bind HCF was also unable to prevent viral IE protein expression.

• Liu Y, Gong W, Huang CC, Herr W, Cheng X
• Crystal structure of the conserved core of the herpes simplex virus transcriptional regulatory protein VP16.
• Genes Dev. 1999; 13: 1692-703
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• On infection, the herpes simplex virus (HSV) virion protein VP16 (Vmw65; alphaTIF) forms a transcriptional regulatory complex-the VP16-induced complex-with two cellular proteins, HCF and Oct-1, on VP16-responsive cis-regulatory elements in HSV immediate-early promoters called TAATGARAT. Comparison of different HSV VP16 sequences reveals a conserved core region that is sufficient for VP16-induced complex formation. The crystal structure of the VP16 core has been determined at 2.1 A resolution. The results reveal a novel, seat-like protein structure. Together with the activity of mutant VP16 proteins, the structure of free VP16 suggests that it contains (1) a disordered carboxy-terminal region that associates with HCF, Oct-1, and DNA in the VP16-induced complex, and (2) a structured region involved in virion assembly and possessing a novel DNA-binding surface that differentiates among TAATGARAT VP16-response elements.

• Hughes TA, La Boissiere S, O'Hare P
• Analysis of functional domains of the host cell factor involved in VP16 complex formation.
• J Biol Chem. 1999; 274: 16437-43
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• We present biochemical analyses of the regions of the host cell factor (HCF) involved in VP16 complex formation and in the association between the N- and C-terminal domains of HCF itself. We show that the kelch repeat region of HCF (residues 1-380) is sufficient for VP16 complex formation, but that residues C-terminal to the repeats (positions 381-450) interfere with this activity. However, these latter residues are required for the interaction between the N- and C-terminal regions of HCF. The extreme C-terminal region of HCF, corresponding to an area of strong conservation with a Caenorhabditis elegans homologue, is sufficient for interaction with the N-terminal region. These results are discussed with respect to possible differences in the roles of HCF in VP16 activity versus its normal cellular function.

• Spatz AM, Ali SA, Auer M, Graf C, Eibl MM, Steinkasserer A
• High level expression and structural characterisation of herpes simplex virus type I transcriptional activator VP16 (alpha-trans inducing factor).
• Biochem Biophys Res Commun. 1998; 251: 235-8
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• We have developed a baculovirus expression system for the rapid and efficient production of large quantities (>10 mg/l) of VP16. The recombinant VP16 binds to a complex of host cell transcription factors and TAATGARAT motif. Secondary structure calculations from circular dichroism measurements indicate a content of 32.0 % alpha-helix and 17.5 % beta-sheet. This is the first structural CD analysis of VP16 which will be very useful for high-throughput assay development and mechanistic studies.

• LaBoissiere S, Walker S, O'Hare P
• Concerted activity of host cell factor subregions in promoting stable VP16 complex assembly and preventing interference by the acidic activation domain.
• Mol Cell Biol. 1997; 17: 7108-18
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• In contrast to our understanding of the roles of Oct-1 and VP16 in VP16-mediated transcriptional activation, virtually nothing is known of the role of the second cellular component, termed host cell factor (HCF), or of its structure-function relationships. We show that the majority of the internal region of HCF, including the repeats involved in HCF cleavage, is dispensable for complex assembly with VP16 and Oct-1. The N-terminal domain of HCF (HCF.N) had only weak VP16 binding and complex promoting activity, while the C-terminal region (HCF.C) had no intrinsic activity. However, the C-terminal region strongly enhanced complex formation and reduced dissociation kinetics when linked to the N-terminal domain (HCF.NC). The potent activity of the HCF.NC fusion in complex assembly was recapitulated in vivo in yeast and mammalian cells. Moreover, HCF.N could promote increased complex formation when the acidic activation domain of VP16 was deleted. Restoration of the activation domain strongly inhibited complex formation with HCF.N, but the addition of the C-terminal domain of HCF restored strong stable complex formation with intact VP16. The results indicate that this C-terminal domain is critically required to alter the presentation of the acidic domain of VP16. Additional results are consistent with the interpretation that this alteration in acidic domain presentation for complex assembly also facilitates the activation function in VP16. The sequence of an HCF homolog from Caenorhabditis elegans shows it to be a natural HCF.NC construct, reinforcing the conclusions from our functional analysis.

• Hagmann M, Georgiev O, Schaffner W
• The VP16 paradox: herpes simplex virus VP16 contains a long-range activation domain but within the natural multiprotein complex activates only from promoter-proximal positions.
• J Virol. 1997; 71: 5952-62
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• Removal of core promoter elements like the TATA box converts several regulatory upstream regions of viral and cellular genes into classical enhancers, i.e., cis-regulatory elements capable of activating transcription over long distances in an orientation-independent manner. This is not the case with herpes simplex virus (HSV) immediate-early gene promoters, which are strongly induced by the viral transactivator VP16 (Vmw65, alphaTIF, ICP25) complexed with the cellular factors Oct-1 and HCF. Here we report that the VP16 complex can readily bring about strong activation from a promoter-proximal position but fails to induce transcription from a distal downstream enhancer position. This is in striking contrast to results obtained with GAL fusion proteins: in this context, the C-terminal "general" activation domain of VP16 activates transcription to high levels over long distances. Thus, this paradoxical behavior suggests that the VP16 activation domain is not accessible to the transcription machinery when the VP16-Oct-1-HCF complex is bound in a remote position. Only upon specific interactions in a promoter-proximal position, perhaps with the basal transcription factors, can transcription be strongly induced. In agreement with such a proposed mechanism, VP16 proteins to which a heterologous general activation domain has been added strongly activate transcription from a downstream position. The biological role of this unexpected and sophisticated mechanism is most probably a limitation of the VP16 activity to the associated immediate-early genes, without undesired long-range effects on other viral promoters within the tightly packed HSV genome.

• Schmelter J, Knez J, Smiley JR, Capone JP
• Identification and characterization of a small modular domain in the herpes simplex virus host shutoff protein sufficient for interaction with VP16.
• J Virol. 1996; 70: 2124-31
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• The herpes simplex virus transactivator VP16 and the virion host shutoff protein vhs are viral structural components that direct the activation of immediate-early gene expression and the arrest of host protein synthesis, respectively, during an infection. Recent studies show that VP16 and vhs physically interact with each other in vitro and in infected cells, suggesting that their respective regulatory functions are coupled. In this report, we used the yeast two-hybrid system and affinity chromatography with purified VP16 fusion proteins to precisely map a region in vhs that directs interaction with VP16. Deletion analysis of vhs demonstrated that a 21-amino-acid-long domain spanning residues 310 to 330 (PAAGGTEMRVSWTEILTQQIA) was sufficient for directing complex formation with VP16 in vivo and in vitro when fused to a heterologous protein. Site-directed mutagenesis of this region identified tryptophan 321 as a crucial determinant for interaction with VP16 in vitro and in vivo and additional residues that are important for stable complex formation in vitro. These findings indicate that vhs residues 310 to 330 constitute an independent and modular binding interface that is recognized by VP16.

• Lam Q et al.
• Herpes simplex virus VP16 rescues viral mRNA from destruction by the virion host shutoff function.
• EMBO J. 1996; 15: 2575-81
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• Herpes simplex virus (HSV) virions contain two regulatory proteins that facilitate the onset of the lytic cycle: VP16 activates transcription of the viral immediate-early genes, and vhs triggers shutoff of host protein synthesis and accelerated turnover of cellular and viral mRNAs. VP16 and vhs form a complex in infected cells, raising the possibility of a regulatory link between them. Here we show that viral protein synthesis and mRNA levels undergo a severe decline at intermediate times after infection with a VP16 null mutant, culminating in virtually complete translational arrest. This phenotype was rescued by a transcriptionally incompetent derivative of VP16 that retains vhs binding activity, and was eliminated by inactivating the vhs gene. These results indicate that VP16 dampens vhs activity, allowing HSV mRNAs to persist in infected cells. Further evidence supporting this hypothesis came from the demonstration that a stably transfected cell line expressing VP16 was resistant to host shutoff induced by superinfecting HSV virions. Thus, in addition to its well known function as a transcriptional activator, VP16 stimulates viral gene expression at a post-transcriptional level, by sparing viral mRNAs from degradation by one of the virus-induced host shutoff mechanisms.

• Misra V, Walker S, Hayes S, O'Hare P
• The bovine herpesvirus alpha gene trans-inducing factor activates transcription by mechanisms different from those of its herpes simplex virus type 1 counterpart VP16.
• J Virol. 1995; 69: 5209-16
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• In herpes simplex virus (HSV)-infected cells, viral gene expression is initiated when the immediate-early, or alpha, genes are transactivated by the alpha gene trans-inducing factor (alpha TIF), a component of the infecting virion. The protein binds to one or more recognition elements (TAATGARAT) in the promoters of alpha genes via interaction with the cellular proteins Oct-1 and CFF. The alpha TIF of HSV (HSV-alpha TIF) is believed to subsequently accelerate the assembly of the transcription complex by direct contact between its carboxyl-terminal acidic activation domain and at least two components of the transcription apparatus, TAFII40 and TFIIB. Like its HSV counterpart, the alpha TIF of bovine herpesvirus (BHV) (designated BHV-alpha TIF) also transactivates alpha gene promoters and for full activity exhibits a requirement for its extended carboxyl-terminal region. Despite this requirement, there is a notable lack of homology to the carboxyl-terminal acidic activation domain of HSV-alpha TIF. We swapped the amino- and carboxyl-terminal domains of HSV-alpha TIF and BHV-alpha TIF to make chimeric proteins. Using these chimeras, we show that the carboxyl terminus of BHV-alpha TIF is insufficient for transactivation, which requires cooperative determinants in both the amino-terminal and carboxyl-terminal regions of the protein. We have previously shown that the amino-terminal determinant in BHV-alpha TIF displays reduced but significant independent transactivation potential. Interestingly, this amino-terminal determinant appears not to reside in the HSV-alpha TIF, which displays no independent amino-terminal activity. Furthermore, we show that the amino-terminal activation domain of BHV-alpha TIF may be able to act synergistically with the carboxyl-terminal activation domain of HSV-alpha TIF, since a chimeric protein containing both domains appeared to be more efficient at activating transcription than either alpha TIF. In addition, the amino terminus of HSV-alpha TIF could not restore activity when linked to the carboxyl terminus of BHV-alpha TIF, while the amino terminus of BHV-alpha TIF reconstituted an intact protein with potent activation potential. We also show that in fusions with the DNA binding domain of GAL4, full activity requires the entire BHV-alpha TIF, although both amino and carboxyl termini display some activity on their own. In contrast, for HSV-alpha TIF, the carboxyl terminus is sufficient and possibly even more potent than the entire protein, while the amino-terminus is devoid of activity.(ABSTRACT TRUNCATED AT 400 WORDS)

• Douville P, Hagmann M, Georgiev O, Schaffner W
• Positive and negative regulation at the herpes simplex virus ICP4 and ICP0 TAATGARAT motifs.
• Virology. 1995; 207: 107-16
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• The control of the ICP0 and ICP4 immediate early genes of herpes simplex virus (HSV) can critically determine the course of viral lytic or latent infections. Their promoters contain so-called TAATGARAT motifs that are activated via a multiprotein complex which includes cellular proteins Oct-1 and HCF and the viral activator (VP16 (= Vmw65, alpha TIF). Relative to the ICP4 promoter TAATGAGAT sequence, the ICP0 promoter motif has a 5' extension that includes a full octamer sequence (ATGCTAATGATAT). It seemed possible that this overlapping octamer site might render the ICP0 promoter element more active by allowing tighter binding of the Oct-1/VP16 complex or more vulnerable to repression by other Oct proteins. Our experiments favor the former possibility. On the one hand, the extended ICP0 site shows stronger binding of the Oct-1/VP16 complex compared to the ICP4 site. Moreover, transcription of a reporter gene with multiple ICP0 sites is strongly activated by VP16 in transfected cells. On the other hand, the ICP0 site is largely refractory toward repression by a different Oct factor (N-Oct2 = Brn1) which competes with Oct-1/VP16 for the site. In marked contrast, multiple copies of the conventional TAATGAGAT motif of ICP4 are poorly activated by VP16, and transcription from this site can be completely repressed by N-Oct2. However, inclusion of the neighboring CGGAAR motifs from the ICP4 promoter, which bind factors GABP alpha and beta, results in a strong synergistic activation. This activity, like that of the complete ICP4 promoter, becomes refractory to repression by competing N-Oct2. Thus the standard TAATGARAT motif of ICP4 is by itself less active and more vulnerable to repression than the extended ICP0 motif, and its activation depends upon synergism with neighboring DNA sites and their cognate factors. This difference between the two types of TAATGARAT motifs may allow for a more complex transcriptional regulation by factor combinations.

• Wu TJ, Monokian G, Mark DF, Wobbe CR
• Transcriptional activation by herpes simplex virus type 1 VP16 in vitro and its inhibition by oligopeptides.
• Mol Cell Biol. 1994; 14: 3484-93
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• VP16 is a herpes simplex virus (HSV)-encoded transcriptional activator protein that is essential for efficient viral replication and as such may be a target for novel therapeutic agents directed against viral gene expression. We have reconstituted transcriptional activation by VP16 in an in vitro system that is dependent on DNA sequences from HSV immediate-early gene promoters and on protein-protein interactions between VP16 and Oct-1 that are required for VP16 activation in vivo. Activation increased synergistically with the number of TAATGARAT elements (the cis-acting element for VP16 activation in vivo) upstream of the core promoter, and mutations of this element that reduce Oct-1 or VP16 DNA binding reduced transactivation in vitro. A VP16 insertion mutant unable to interact with Oct-1 was inactive, but, surprisingly, a deletion mutant lacking the activation domain was approximately 65% as active as the full-length protein. The activation domains of Oct-1 were necessary for activation in reactions containing the VP16 deletion mutant, and they contributed significantly to activation by full-length VP16. Addition of a GA-rich element present in many HSV immediate-early gene enhancers synergistically stimulated VP16-activated transcription. Finally, oligopeptides that are derived from a region of VP16 thought to contact a cellular factor known as HCF (host cell factor) and that inhibit efficient VP16 binding to the TAATGARAT element also specifically inhibited VP16-activated, but not basal, transcription. Amino acid substitutions in one of these peptides identified three residues that are absolutely required for inhibition and presumably for interaction of VP16 with HCF.

• Regier JL, Shen F, Triezenberg SJ
• Pattern of aromatic and hydrophobic amino acids critical for one of two subdomains of the VP16 transcriptional activator.
• Proc Natl Acad Sci U S A. 1993; 90: 883-7
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• Structural features of the transcriptional activation domain of the herpes simplex virion protein VP16 were examined by oligonucleotide-directed mutagenesis. Extensive mutagenesis at position 442 of the truncated VP16 activation domain (delta 456), normally occupied by a phenylalanine residue, demonstrated the importance of an aromatic amino acid at that position. On the basis of an alignment of the VP16 sequence surrounding Phe-442 and the sequences of other transcriptional activation domains, we subjected leucine residues at positions 439 and 444 of VP16 to mutagenesis. Results from these experiments suggest that bulky hydrophobic residues flanking Phe-442 also contribute significantly to the function of the truncated VP16 activation domain. Restoration of amino acids 457-490 to various Phe-442 mutants partially restored activity. Although the pattern of amino acids surrounding Phe-473 resembles that surrounding Phe-442, mutations of Phe-473 did not dramatically affect activity; in fact, Phe-475 appears more sensitive to mutations than does Phe-473. We infer that the two regions of VP16 (amino acids 413-456 and 457-490) possess unique structural features, although neither is likely to be an amphipathic alpha-helix or an "acidic blob." These results, considered with previous in vitro activation and inhibition studies, suggest that the two subdomains of VP16 affect transcription by different mechanisms.

• Hayes S, O'Hare P
• Mapping of a major surface-exposed site in herpes simplex virus protein Vmw65 to a region of direct interaction in a transcription complex assembly.
• J Virol. 1993; 67: 852-62
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• The cellular factor Oct-1 is selectively recruited, together with at least one other cellular protein (CFF), into a multicomponent transcription complex whose assembly is directed by the herpes simplex virus regulatory protein Vmw65 (VP16). The acidic carboxy terminus of Vmw65 is not involved in assembly of the complex but is absolutely required for subsequent transcriptional activation. Elucidation of the mechanism of action of Vmw65 is important for an understanding not only of combinatorial control of gene expression by POU- and homeodomain proteins but also of the interaction(s) between activation domains of regulatory proteins and components of the basal transcriptional apparatus. We used a combination of limited proteolysis with a number of site-specific proteases and immunological detection to demonstrate the presence of two main surface-exposed regions in Vmw65. We mapped these sites to within a few amino acids at positions 365-370 408/409. The site at 408/409 is indicative of a flexible exposed linker region between the acidic carboxy-terminal activation domain (residues 430-480) and an N-terminal domain involved in complex formation with the two cellular factors. The site around residues 365-370 is precisely within a region that results from this and other laboratories have shown to be critical for complex formation. Furthermore, we show that this site is selectively protected from proteolysis after complex assembly. Finally, using a series of overlapping peptide encompassing this region, we show that the eight amino acids, R-E-H-A-Y-S-R-A, from positions 360 through 367 are sufficient to inhibit complex formation by intact Vmw65. We propose that these residues contain sufficient information to selectively bind one of the cellular partners involved in complex assembly and that these residues are located in a physical surface-exposed domain of the protein.

• Lillycrop KA, Estridge JK, Latchman DS
• The octamer binding protein Oct-2 inhibits transactivation of the herpes simplex virus immediate-early genes by the virion protein Vmw65.
• Virology. 1993; 196: 888-91
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• Transactivation by a complex of the cellular transcription factor Oct-1 and the virion protein Vmw65 is necessary for the high-level activity of the HSV immediate-early promoters during lytic infection. We show that this trans-activation can be inhibited by two forms of the Oct-2 transcription factor which are expressed at high levels in neuronal cells as well as by the isolated DNA binding, POU domain of Oct-2. The inhibition of Oct-1-Vmw65 DNA binding by these neuronal forms of Oct-2 is likely to play a critical role in the nonpermissivity of neuronal cells for the HSV lytic cycle and therefore in the establishment of latent infections.

• Wilson AC, Cleary MA, Lai JS, LaMarco K, Peterson MG, Herr W
• Combinatorial control of transcription: the herpes simplex virus VP16-induced complex.
• Cold Spring Harb Symp Quant Biol. 1993; 58: 167-78

• Wilson AC, LaMarco K, Peterson MG, Herr W
• The VP16 accessory protein HCF is a family of polypeptides processed from a large precursor protein.
• Cell. 1993; 74: 115-25
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• Upon lytic infection of permissive cells, the herpes simplex virus (HSV) transactivator protein VP16 associates with an accessory protein termed host cell factor (HCF). Binding to HCF activates VP16 for association with the octamer motif-binding protein Oct-1, to form a multiprotein-DNA complex responsible for activating transcription of the HSV immediate early genes. We show that HCF comprises a series of related polypeptides that range from 110 to 300 kd, all of which are encoded by a single gene. Although there is no obvious sequence similarity between HCF and other known proteins, HCF contains eight repeats of a new 26 amino acid motif. cDNAs encoding HCF predict a large open reading frame of 2035 codons. When expressed in human cells, this large open reading frame encodes both the 300 kd and smaller HCF polypeptides, indicating that the smaller polypeptides arise by processing of the 300 kd protein.

• Cress WD, Triezenberg SJ
• Critical structural elements of the VP16 transcriptional activation domain.
• Science. 1991; 251: 87-90
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• Virion protein 16 (VP16) of herpes simplex virus type 1 contains an acidic transcriptional activation domain. Missense mutations within this domain have provided insights into the structural elements critical for its function. Net negative charge contributed to, but was not sufficient for, transcriptional activation by VP16. A putative amphipathic alpha helix did not appear to be an important structural component of the activation domain. A phenylalanine residue at position 442 was exquisitely sensitive to mutation. Transcriptional activators of several classes contain hydrophobic amino acids arranged in patterns resembling that of VP16. Therefore, the mechanism of transcriptional activation by VP16 and other proteins may involve both ionic and specific hydrophobic interactions with target molecules.

• Latchman DS
• The herpes simplex virus virion protein Vmw65 transcriptionally activates the gene encoding the U4 snRNA but not that encoding the U2 snRNA during lytic infection.
• Biochem J. 1991; 275: 369-72
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• Although lytic infection with herpes simplex virus (HSV) causes the repression of most host cell biosynthesis, it results in increased transcription of the cellular gene encoding the U4 snRNA, leading to accumulation of this snRNA. In contrast, no increased transcription of the gene encoding the U2 snRNA or accumulation of this RNA is observed in infected cells. These effects are mediated by the HSV virion protein Vmw65, which activates the U4 gene but does not affect the U2 gene. The significance of this difference between the U2 and U4 genes is discussed with regard to the presence in both of these genes of an identical octamer-binding site for the cellular transcription factor Oct-1 which complexes with Vmw65.

• Stern S, Herr W
• The herpes simplex virus trans-activator VP16 recognizes the Oct-1 homeo domain: evidence for a homeo domain recognition subdomain.
• Genes Dev. 1991; 5: 2555-66
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• The homeo domain of the Oct-1 transcription factor directs formation of a multiprotein-DNA complex containing Oct-1, the herpes simplex virus (HSV) trans-activator VP16, and a second host cell factor (HCF). This VP16-induced complex alters the regulatory activity of Oct-1, in part, by associating it with the potent VP16 acidic transcriptional activation domain. Here, we show that in the absence of HCF, VP16 can recognize specifically the Oct-1 homeo domain. A region of VP16 near the acidic activation domain appears to be involved exclusively in homeo domain recognition because a 4-amino-acid insertion within this region only affects the ability of VP16 to interact with Oct-1, leaving its DNA- and HCF-binding activities unchanged. A 33-amino-acid peptide containing this region complexes with the Oct-1 POU domain bound to DNA, suggesting that this VP16 region contains an autonomous homeo domain recognition subdomain.