Matthew S. Wiebe

Identification of the Transactivation Domain of the Transcription Factor Sox-2 and an Associated Co-activator

The importance of interactions between Sox and POU transcription factors in the regulation of gene expression is becoming increasingly apparent. Recently, many examples of the involvement of Sox-POU partnerships in transcription have been discovered, including a partnership between Sox-2 and Oct-3. Little is known about the mechanisms by which these factors modulate transcription. To better understand the molecular interactions involved, we mapped the location of the transactivation domain of Sox-2. This was done in the context of its interaction with Oct-3, as well as its ability to transactivate as a fusion protein linked to the DNA-binding domain of Gal4. Both approaches demonstrated that Sox-2 contains a transactivation domain in its C-terminal half, containing a serine-rich region and the C terminus. We also determined that the viral oncoprotein E1a inhibits the ability of the Gal4/Sox-2 fusion protein to transactivate, as well as the transcriptional activation mediated by the combined action of Sox-2 and Oct-3. In contrast, a mutant form of E1a, unable to bind p300, lacks both of these effects. Importantly, we determined that p300 overcomes the inhibitory effects of E1a in both assays. Together, these findings suggest that Sox-2 mediates its effects, at least in part, through the co-activator p300.

Identification of Novel Domains within Sox-2 and Sox-11 Involved in Autoinhibition of DNA Binding and Partnership Specificity

Sox transcription factors play key regulatory roles throughout development, binding DNA through a consensus (A/T)(A/T)CAA(A/T)G sequence. Although many different Sox proteins bind to this sequence, it has been observed that gene regulatory elements are commonly responsive to only a small subset of the entire family, implying that regulatory mechanisms exist to permit selective DNA binding and/or transactivation by Sox family members. To identify and explore the mechanisms modulating gene activation by Sox proteins further, we compared the function of Sox-2 and Sox-11. This led to the discovery that Sox proteins are regulated differentially at multiple levels, including transactivation, protein partnerships with Pit-Oct-Unc (POU) transcription factors, and DNA binding autoregulation. Specifically, we determined that Sox-11 activates transcription more strongly than Sox-2 and that the transactivation domain of Sox-11 is primarily responsible for this capability. Additionally, we demonstrate that the Sox-11 DNA binding domain is responsible for selective cooperation with the POU factor Brn-2. This requirement cannot be replaced by the DNA binding domain of Sox-2, indicating that the DNA binding domain of Sox proteins is critical for Sox-POU partnerships. Interestingly, we have also determined that a conserved domain of Sox-11 has the novel capability of autoinhibiting its ability to bind DNA in vitro and to activate gene expression in vivo. Our findings suggest that the autoinhibitory domain can repress promiscuous binding of Sox-11 to DNA and plays an important role in regulating the recruitment of Sox-11 to specific genes.

Mice Deficient in the Serine/Threonine Protein Kinase VRK1 Are Infertile Due to a Progressive Loss of Spermatogonia

Abstract The VRK1 protein kinase has been implicated as a pro-proliferative factor. Genetic analyses of mutant alleles of the Drosophila and Caenorhabditis elegans VRK1 homologs have revealed phenotypes ranging from embryonic lethality to mitotic and meiotic defects with resultant sterility. Herein, we describe the first genetic analysis of murine VRK1. Two lines of mice containing distinct gene-trap integrations into the Vrk1 locus were established. Insertion into intron 12 (GT12) spared VRK1 function, enabling the examination of VRK1 expression in situ. Insertion into intron 3 (GT3) disrupted VRK1 function, but incomplete splicing to the gene trap rendered this allele hypomorphic (∼15% of wild-type levels of VRK1 remain). GT3/GT3 mice are viable, but both males and females are infertile. In testes, VRK1 is expressed in Sertoli cells and spermatogonia. The infertility of GT3/GT3 male mice results from a progressive defect in spermatogonial proliferation or differentiation, culminating in the absence of mitotic and meiotic cells in adult testis. These data demonstrate an important role for VRK1 in cell proliferation and confirm that the need for VRK1 during gametogenesis is evolutionarily conserved.

Barrier to Autointegration Factor (BANF1): interwoven roles in nuclear structure, genome integrity, innate immunity, stress responses and progeria.

The Barrier to Autointegration Factor (BAF or BANF1) is an abundant, highly conserved DNA binding protein. BAF is involved in multiple pathways including mitosis, nuclear assembly, viral infection, chromatin and gene regulation and the DNA damage response. BAF is also essential for early development in metazoans and relevant to human physiology; BANF1 mutations cause a progeroid syndrome, placing BAF within the laminopathy disease spectrum. This review summarizes previous knowledge about BAF in the context of recent discoveries about its protein partners, posttranslational regulation, dynamic subcellular localizations and roles in disease, innate immunity, transposable elements and genome integrity.

Molecular Characterization of the Host Defense Activity of the Barrier to Autointegration Factor against Vaccinia Virus

ABSTRACT The barrier to autointegration factor (BAF) is an essential cellular protein with functions in mitotic nuclear reassembly, retroviral preintegration complex stability, and transcriptional regulation. Molecular properties of BAF include the ability to bind double-stranded DNA in a sequence-independent manner, homodimerize, and bind proteins containing a LEM domain. These capabilities allow BAF to compact DNA and assemble higher-order nucleoprotein complexes, the nature of which is poorly understood. Recently, it was revealed that BAF also acts as a potent host defense against poxviral DNA replication in the cytoplasm. Here, we extend these observations by examining the molecular mechanism through which BAF acts as a host defense against vaccinia virus replication and cytoplasmic DNA in general. Interestingly, BAF rapidly relocalizes to transfected DNA from a variety of sources, demonstrating that BAFs activity as a host defense factor is not limited to poxviral infection. BAFs relocalization to cytoplasmic foreign DNA is highly dependent upon its DNA binding and dimerization properties but does not appear to require its LEM domain binding activity. However, the LEM domain protein emerin is recruited to cytoplasmic DNA in a BAF-dependent manner during both transfection and vaccinia virus infection. Finally, we demonstrate that the DNA binding and dimerization capabilities of BAF are essential for its function as an antipoxviral effector, while the presence of emerin is not required. Together, these data provide further mechanistic insight into which of BAFs molecular properties are employed by cells to impair the replication of poxviruses or respond to foreign DNA in general.

NF-Y Behaves as a Bifunctional Transcription Factor That Can Stimulate or Repress the FGF-4 Promoter in an Enhancer-Dependent Manner

NF-Y is a bifunctional transcription factor capable of activating or repressing transcription. NF-Y specifically recognizes CCAAT box motifs present in many eukaryotic promoters. The mechanisms involved in regulating its activity are poorly understood. Previous studies have shown that the FGF-4 promoter is regulated positively by its CCAAT box and NF-Y in embryonal carcinoma (EC) cells where the distal enhancer of the FGF-4 gene is active. Here, we demonstrate that the CCAAT box functions as a negative cis-regulatory element when cis-regulatory elements of the FGF-4 enhancer are disrupted, or after EC cells differentiate and the FGF-4 enhancer is inactivated. We also demonstrate that NF-Y mediates the repression of the CCAAT box and that NF-Y associates with the endogenous FGF-4 gene in both EC cells and EC-differentiated cells. Importantly, we also determined that the orientation and the position of the CCAAT box are critical for its role in regulating the FGF-4 promoter. Together, these studies demonstrate that the distal enhancer of the FGF-4 gene determines whether the CCAAT box of the FGF-4 promoter functions as a positive or a negative cis-regulatory element. In addition, these studies are consistent with NF-Y playing an architectural role in its regulation of the FGF-4 promoter.

Molecular Genetic and Biochemical Characterization of the Vaccinia Virus I3 Protein, the Replicative Single-Stranded DNA Binding Protein

ABSTRACT Vaccinia virus, the prototypic poxvirus, efficiently and faithfully replicates its ∼200-kb DNA genome within the cytoplasm of infected cells. This intracellular localization dictates that vaccinia virus encodes most, if not all, of its own DNA replication machinery. Included in the repertoire of viral replication proteins is the I3 protein, which binds to single-stranded DNA (ssDNA) with great specificity and stability and has been presumed to be the replicative ssDNA binding protein (SSB). We substantiate here that I3 colocalizes with bromodeoxyuridine (BrdU)-labeled nascent viral genomes and that these genomes accumulate in cytoplasmic factories that are delimited by membranes derived from the endoplasmic reticulum. Moreover, we report on a structure/function analysis of I3 involving the isolation and characterization of 10 clustered charge-to-alanine mutants. These mutants were analyzed for their biochemical properties (self-interaction and DNA binding) and biological competence. Three of the mutant proteins, encoded by the I3 alleles I3-4, -5, and -7, were deficient in self-interaction and unable to support virus viability, strongly suggesting that the multimerization of I3 is biologically significant. Mutant I3-5 was also deficient in DNA binding. Additionally, we demonstrate that small interfering RNA (siRNA)-mediated depletion of I3 causes a significant decrease in the accumulation of progeny genomes and that this reduction diminishes the yield of infectious virus.

Barrier to autointegration factor (BAF) inhibits vaccinia virus intermediate transcription in the absence of the viral B1 kinase

Barrier to autointegration factor (BAF/BANF1) is a cellular DNA-binding protein found in the nucleus and cytoplasm. Cytoplasmic BAF binds to foreign DNA and can act as a defense against vaccinia DNA replication. To evade BAF, vaccinia expresses the B1 kinase, which phosphorylates BAF and blocks its ability to bind DNA. Interestingly, B1 is also needed for viral intermediate gene expression via an unknown mechanism. Therefore, we evaluated the impact of B1-BAF signaling on vaccinia transcription. Strikingly, the decrease in vaccinia transcription caused by loss of B1 can be rescued by depletion of BAF. The repressive action of BAF is greatest on a viral promoter, and is more modest when non-vaccinia promoters are employed, which suggests BAF acts in a gene specific manner. These studies expand our understanding of the role of the B1 kinase during infection and provide the first evidence that BAF is a defense against viral gene expression.

Cell- and Virus-Mediated Regulation of the Barrier-to-Autointegration Factor's Phosphorylation State Controls Its DNA Binding, Dimerization, Subcellular Localization, and Antipoxviral Activity

ABSTRACT Barrier-to-autointegration factor (BAF) is a DNA binding protein with multiple cellular functions, including the ability to act as a potent defense against vaccinia virus infection. This antiviral function involves BAFs ability to condense double-stranded DNA and subsequently prevent viral DNA replication. In recent years, it has become increasingly evident that dynamic phosphorylation involving the vaccinia virus B1 kinase and cellular enzymes is likely a key regulator of multiple BAF functions; however, the precise mechanisms are poorly understood. Here we analyzed how phosphorylation impacts BAFs DNA binding, subcellular localization, dimerization, and antipoxviral activity through the characterization of BAF phosphomimetic and unphosphorylatable mutants. Our studies demonstrate that increased phosphorylation enhances BAFs mobilization from the nucleus to the cytosol, while dephosphorylation restricts BAF to the nucleus. Phosphorylation also impairs both BAFs dimerization and its DNA binding activity. Furthermore, our studies of BAFs antiviral activity revealed that hyperphosphorylated BAF is unable to suppress viral DNA replication or virus production. Interestingly, the unphosphorylatable BAF mutant, which is capable of binding DNA but localizes predominantly to the nucleus, was also incapable of suppressing viral replication. Thus, both DNA binding and localization are important determinants of BAFs antiviral function. Finally, our examination of how phosphatases are involved in regulating BAF revealed that PP2A dephosphorylates BAF during vaccinia infection, thus counterbalancing the activity of the B1 kinase. Altogether, these data demonstrate that phosphoregulation of BAF by viral and cellular enzymes modulates this protein at multiple molecular levels, thus determining its effectiveness as an antiviral factor and likely other functions as well. IMPORTANCE The barrier-to-autointegration factor (BAF) contributes to cellular genomic integrity in multiple ways, the best characterized of which are as a host defense against cytoplasmic DNA and as a regulator of mitotic nuclear reassembly. Although dynamic phosphorylation involving both viral and cellular enzymes is likely a key regulator of multiple BAF functions, the precise mechanisms involved are poorly understood. Here we demonstrate that phosphorylation coordinately regulates BAFs DNA binding, subcellular localization, dimerization, and antipoxviral activity. Overall, our findings provide new insights into how phosphoregulation of BAF modulates this protein at multiple levels and governs its effectiveness as an antiviral factor against foreign DNA.

Bovine herpesvirus 1 productive infection stimulates inflammasome formation and caspase 1 activity.

Bovine herpesvirus 1 (BoHV-1), a significant viral pathogen of cattle, causes inflammation in affected tissue during acute infection. Consequently, we tested whether productively infected bovine cells stimulate inflammasome formation. Expression of two components required for inflammasome formation, the DNA sensor IFI16 (gamma-interferon-inducible protein 16) and NLRP3 (NOD-like receptor family, pyrin domain containing 3), were induced in bovine kidney cells by eight hours after infection. IFI16 was detected in punctate granules localized to the cytoplasm and nucleus. During productive infection, more than ten times more cells were caspase 1 positive, which is activated following inflammasome formation. Two caspase 1 inhibitors had no effect on productive infection. Conversely, another caspase 1 inhibitor, glyburide, significantly inhibited virus infection suggesting it had off-target effects on related enzymes or interfered with infection via non-enzymatic mechanisms. Collectively, these studies demonstrated that BoHV-1 infection stimulated inflammasome formation, which we predict is important for clinical symptoms in cattle.